Organotellurium compounds, compositions and methods of use thereof

ABSTRACT

A compound of formula (I) as described herein and methods and uses thereof as probes in the mass tagging of biosensors or biologically active materials for use in mass cytometry analysis of tissue samples such as in the detection, labelling and quantification of oxygen-deprived cells by using, for example, tellurophene-tagged 2-nitroimidazole.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a Patent Cooperation Treat Application which claims the benefitof 35 U.S.C. §119 based on the priority of U.S. Provisional PatentApplication No. 62/039,762, filed Aug. 20, 2014 and U.S. ProvisionalPatent Application No. 62/165,002, filed May 21, 2015, each of which areincorporated herein by reference in their entirety.

FIELD

The application pertains to organotellurophene compounds andparticularly to organotellurophene probes for mass cytometry.

BACKGROUND

Characterization of single cells in tissue samples requires a highlyparameterized assay.¹ Fluorescence-based flow cytometry (FC) has beenthe method of choice to study heterogeneous cell populations as itallows for 5-10 parameters to be routinely analyzed.² However, FC cannotbe used for highly parameterized assays (>20 parameters) due to thespectral overlap of the fluorophores used for analyte detection.³ Asolution to this problem is to substitute the optical detection andfluorescently tagged antibodies in FC, for mass detection with aninductively-coupled plasma spectrometer (ICP-MS) and isotope-taggedantibodies. This technology, known as mass cytometry (MC), is capable ofdetecting numerous bioorthogonal isotopes (theoretically >100) withsingle mass unit resolution over multiple orders of magnitude.¹ MCallows experiments analogous to flow cytometry but with significantlygreater parameterization. MC has been used to detect and quantify 34cellular parameters simultaneously to reveal the drug response across ahuman hematopoietic continuum.⁴

MC experiments can be done by using commercially available MaxPar© tolabel antibodies with metal chelating polymers that bind a range of highmolecular weight metal isotopes, usually lanthanides. Element tagsattached to polymer backbones are described in U.S. Pat. No. 9,012,239.Specific examples disclosed include elemental tags comprising the metalchelating groups diethylenetriaminepentaacetate (DTPA) and1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

Other mass tagged reagents are desirable.

The first organotellurium compound was synthesized by Wohler in 1840.⁶Increasingly organotellurium compounds are being investigated in livingsystems, although this area of research remains underdeveloped.^(7,8)Tellurium has no known biological role in prokaryotic or eukaryoticcells. In biological systems, tellurium metabolism is poorly understood,however it is presumed to follow the metabolic pathway of its analogue,selenium. Microorganisms have been found to methylate inorganictellurium to volatile or ionic species for excretion. Experimentalevidence of this process is scarce due to the instability of themetabolites⁷. However the number reports of cellular studies involvingaryl, vinylic, alkynyl and alkyl telluroethers in biological systems areincreasing.⁹⁻¹⁴ The majority of this research has been based upon theability of aryl telluroethers to mimic glutathione peroxidase activityproviding, in some cases, resistance to oxidative stress and in othercase disregulating redox homeostatsis leading to appoptosis.^(15,16)Recent murine studies have shown diverse effects from the expectedtoxicity of an amino acid based aryl telluroether to increased memory inmice treated with an alkyl telluroether.^(7,18)

SUMMARY

Mass Cytometry (MC) probes that can, in an embodiment, be used to assaycellular biochemistry are described herein. FIG. 1 depicts an embodimentof an MC probe. Ideally, the mass tag should be accessible in a highyielding synthesis amenable to isotope incorporation, be stable underbiologically-relevant conditions and have low toxicity. An MC-probe(Telox) for measuring cellular hypoxia was constructed (described inU.S. application Ser. No. 62/039,762).⁵ This probe used a2-nitroimidazole as the activity group for hypoxic-specific labellingand a methyl telluroether functionality as the tag unit for MCdetection.⁵ Tellurium was chosen to be the element for detection as itis known to form stable bonds with carbon and it has 8 naturallyoccurring isotopes that can be accessed to generate a series of uniquelyidentifiable, biologically indistinguishable MC probes using the samechemistry.

In Telox, the telluroether functionality had moderate stability and ametabolic LD₅₀ value close to the required assay concentration.

Described herein are the synthesis, aqueous/aerobic stability and invitro toxicity of a series of alkyl telluroethers and tellurophenefunctional groups.

Other features and advantages of the present application will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating embodiments of the application, are given byway of illustration only and the scope of the claims should not belimited by these embodiments, but should be given the broadestinterpretation consistent with the description as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present disclosure will now be described in relationto the drawings in which:

FIG. 1 shows the general requirements and design of a mass cytometryprobe in certain embodiments of the present application.

FIG. 2 shows the results of ¹H NMR stability experiments for exemplarycompounds 1-11. (A) Compounds 1, 2 and 7. (B) Compounds 3, 4, 5 and 6.(C) Compounds 8, 9, 10 and 11. The organotellurium compounds (˜150 μM)were dissolved in d-DMSO with 1,3,5-trioxane, the secondary internalstandard. The compounds were kept in clear glass 20 mL vials. The vialswere kept in a moisture free environment for 24 hours with continuoussupply of ambient atmosphere dried using a series of bubblers containingphosphoric acid, potassium hydroxide and calcium sulfate. Aliquots wereanalyzed by ¹H NMR and the organotellurium signals and the d5-H-DMSOpeaks were integrated. The ratio between the DMSO and theorganotellurium protons at time 0 were taken and normalized to generatea degradation plot. Experimental error was calculated by generatingtriplicate integration data from each individual preliminary NMR data.This error takes into consideration of integration bias and instrumentfluctuations.

FIG. 3 shows the results of degradation NMR experiments for exemplarycompounds 10 and 11 in 50% d-DMSO, d-PBS buffer solutions. The ¹⁹F NMRof compound and ¹H NMR of compound 11 were taken at shown time points.Compound 10 used trifluoroacetic acid as the internal standard andcompound 11 used d5-DMSO.

FIG. 4: Schematic representation of Telox/Telox2 cell labelling foranalysis via mass cytometry on a second-generation CyTOF instrument. b)Density map of signal event length vs. ¹³⁰Te signal (arbitrary units).c) Density plot output from the top-right gate in b) of ¹⁹³Ir signal(arbitrary units) vs. ¹⁰³Rh signal (arbitrary units). More than 93% ofdetected events fall in the square gate. d) Density plot output from thebottom-left gate in b) of ¹⁹³Ir signal (arbitrary units) vs. ¹⁰³Rhsignal (arbitrary units). More than 89% of detected events fall in thesquare gate. e) Population histograms of cell ¹³⁰Te content (arbitraryunits). Oxygen concentrations are listed as numerical percentages,P=Pimonidazole (100 μM). Blue and red histograms arePimonidazole-negative controls. Orange and green histograms arePimonidazole-positive competition experiments. Note: warmer colors indensity plots indicate higher cell population density.

FIG. 5 shows the stability of an exemplary compound, Telox-2, as afunction of time as monitored by ¹H NMR. Concentration of Telox-2=200 μM(0.01% D₆-DMSO in D₂O).

FIG. 6 shows the stability of exemplary compound, Telox-2, as a functionof time as monitored by ultraviolet-visible spectroscopy. Concentrationof Telox-2=200 μM (0.01% DMSO in phosphate-buffered saline). The smallspectrum identifies the unique absorptions of the tellurophene andnitroimidazole functionalities.

FIG. 7 shows a) the cell proliferation rate of HCT116 cells as afunction of time in the presence of various concentrations of theexemplary compound, Telox-2 (confluency analysis); and b) the metabolictoxicity of the exemplary compound, Telox-2, in Jurkat cells as measuredby reduction of WST-1. Cells were incubated with Telox-2 for 24 hoursprior to a 30 minute exposure to WST-1.

FIG. 8 shows a) the absolute ¹³⁰Te signal as a function of concentrationas determined by mass cytometry analysis of HCT116 cells incubated withthe exemplary compound, Telox-2, for 3 hours in either near-anoxic (˜0%O₂), hypoxic (˜1% O₂) or normoxic (21% O₂) atmosphere; and b) thesignal-to-noise (fold-change) representation of the data presented inpart a.

FIG. 9 shows a) Absolute ¹³⁰Te signal as a function of time asdetermined by mass cytometry analysis of HCT116 cells incubated with 10μM of the exemplary compound, Telox-2 (constant exposure), in eithernear-anoxic (˜0% O₂), hypoxic (˜1% O₂) or normoxic (21% O₂) atmosphere;b) the signal-to-noise (fold-change) representation of the datapresented in part a; and c) the absolute ¹³⁰Te signal as a function oftime as determined by mass cytometry analysis of HCT116 cells incubatedwith 10 μM Telox-2 for 3 hours followed by replacement ofTelox-2-containing media with fresh media in either near-anoxic (˜0%O₂), hypoxic (˜1% O₂) or normoxic (21% O₂) atmosphere; and d) thesignal-to-noise (fold-change) representation of the data presented inpart c.

FIG. 10 shows a) the mass cytometry histograms of wild-type HCT116 cellsincubated with 10 μM of the exemplary compound, Telox-2, for 3 hours inatmosphere containing either 21% O₂, 1% O₂, 0.2% O₂, or <0.02% O₂); b)the mass cytometry histograms of mutant HCT116 cells overexpressing PORincubated with 10 μM Telox-2 for 3 hours in atmosphere containing either21% O₂, 1% O₂, 0.2% O₂ or <0.02% O₂; and c) the mass cytometryhistograms of mutant HCT116 cells unable to express POR incubated with10 μM Telox-2 for 3 hours in atmosphere containing either 21% O₂, 1% O₂,0.2% O₂, or <0.02% 02.

FIG. 11 shows the competitive labelling with the exemplary compound,Telox-2, and pimonidazole in HCT116 cells incubated in either normoxic(21% O₂) or near-anoxic (˜0% O₂) atmosphere. Concentration Telox-2=10μM. The absolute ¹³⁰Te signal was measured using mass cytometry.

DETAILED DESCRIPTION OF THE DISCLOSURE I. Definitions

The term “a cell” as used herein includes a single cell as well as aplurality or population of cells.

The term “antibody” as used herein is intended to include monoclonalantibodies, polyclonal antibodies, and chimeric antibodies and bindingfragments thereof. The antibody may be from recombinant sources and/orproduced in transgenic animals. Antibodies can be fragmented usingconventional techniques. For example, F(ab′)2 fragments can be generatedby treating the antibody with pepsin. The resulting F(ab′)2 fragment canbe treated to reduce disulfide bridges to produce Fab′ fragments. Papaindigestion can lead to the formation of Fab fragments. Fab, Fab′ andF(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecificantibody fragments and other fragments can also be synthesized byrecombinant techniques. Antibody fragments as used herein mean bindingfragments

The term “biosensor” as used herein means any enzyme substrate that 1)is converted by an enzyme to reactive products (such as but not limitedto, quinone methide intermediates), insoluble products and/or membranelocalizing products (e.g. fatty acid containing products), wherein saidproducts label a cell (e.g. a cell constituent), the local tissueenvironment or is an irreversible enzyme inhibitor that labels activeenzymes, and 2) can be conjugated to an organotellurophene compound,optionally a compound of formula (I). In some embodiments, the biosensoris coupled to and/or further comprises one or more mass tags or asupporting structure of a mass tag.

The term “biologically active material” as used herein means an entityselected from a cell, virus, subcellular particle, polypeptide, nucleicacid, peptidic nucleic acid, oligosaccharide, polysaccharidelipopolysaccharide, cellular metabolite, hapten, hormone,pharmacologically active substance, alkaloid, steroid, vitamin, aminoacid and sugar, and includes for example synthetic mimetics thereof. Insome embodiments, the biologically active material is coupled to and/orfurther comprises one or more mass tags or a supporting structure of amass tag.

The term “distinct tellurium isotope” as used herein refers to Te atomsin a compound having one or more atoms of a single tellurium isotope.For example, a series of mass tagged entities can be employed in anassay each having a different distinct tellurium isotope, such that eachcompound comprising a distinct tellurium isotope is distinguishable fromother compounds.

The term “distinct mass” as used herein indicates that the compound hasone or more atoms of a single tellurium isotope or a unique combinationof tellurium isotopes alone (e.g. distinct tellurium mass) or incombination with other mass tags. An example includes a series ofcompounds, optionally polymers, each with different levels of differenttellurium isotopes alone or combined with other mess tags, optionallyfor use in barcoding embodiments. Alternatively the compound for examplean enzyme substrate may comprise multiple isotopes of tellurium alone orin combination with other mass tags. Upon cleavage of the substrate, aratiometric approach can be used to assess whether the enzyme is active.

The term “mass tag” as used herein refers to a molecule that comprisesat least one specific elemental isotopic composition that serves todistinguish a molecule to which the tag is attached, or optionally thetag itself, from other molecules comprising a different elementalisotopic composition using a mass spectral analysis. In someembodiments, the mass tag comprises at least one elemental isotope and asupporting structure for the at least one elemental isotope.

The term “metabolic labelling” as used herein refers to incorporation ofa biomolecule into a macromolecule, for example incorporation of anamino acid into a protein, or a monosaccharide into a polysaccharide orglycoprotein.

As used herein “organotellurophene tag” means any tellurophenecontaining compound comprising a tellurophene moiety and a linker (L)that is for example compact and can be conjugated to biosensor, apolymeric backbone and/or a biologically active material and includesfor example organotellurophene compounds described herein and/ordescribed in U.S. application 62/039,762, hereby incorporated byreference. For example, the organotellurophene tag can comprise atellurophene moiety, a linker conjugated to a biosensor such as anantibody either directly or indirectly, optionally indirectly via apolymer backbone.

The term “protease” as used herein is intended to include peptidases andproteinases and is the subset of enzymes that can catalyze the cleavageof a peptide bond and includes for example cysteine proteases, aspartateproteases, metalloproteases and serine proteases.

A “subcellular particle” as used herein includes an organelle, such asnucleus, lysosome, endosome, mitochondria, microsomes and the like.

As used in this application, the words “comprising” (and any form ofcomprising, such as “comprise” and “comprises”), “having” (and any formof having, such as “have” and “has”), “including” (and any form ofincluding, such as “include” and “includes”) or “containing” (and anyform of containing, such as “contain” and “contains”), are inclusive oropen-ended and do not exclude additional, unrecited elements or processsteps.

As used in this application and claim(s), the word “consisting” and itsderivatives, are intended to be close ended terms that specify thepresence of stated features, elements, components, groups, integers,and/or steps, and also exclude the presence of other unstated features,elements, components, groups, integers and/or steps.

The term “consisting essentially of”, as used herein, is intended tospecify the presence of the stated features, elements, components,groups, integers, and/or steps as well as those that do not materiallyaffect the basic and novel characteristic(s) of these features,elements, components, groups, integers, and/or steps.

The terms “about”, “substantially” and “approximately” as used hereinmean a reasonable amount of deviation of the modified term such that theend result is not significantly changed. These terms of degree should beconstrued as including a deviation of at least ±5% of the modified termif this deviation would not negate the meaning of the word it modifies.

The present description refers to a number of chemical and biologicalterms and abbreviations used by those skilled in the art. Nevertheless,definitions of selected terms are provided for clarity and consistency.

The term “suitable” as used herein means that the selection of theparticular compound or conditions would depend on the specific syntheticmanipulation to be performed, and the identity of the molecule(s) to betransformed, but the selection would be well within the skill of aperson trained in the art. All process/method steps described herein areto be conducted under conditions sufficient to provide the productshown. A person skilled in the art would understand that all reactionconditions, including, for example, reaction solvent, reaction time,reaction temperature, reaction pressure, reactant ratio and whether ornot the reaction should be performed under an anhydrous or inertatmosphere, can be varied to optimize the yield of the desired productand it is within their skill to do so.

As used in this application, the singular forms “a”, “an” and “the”include plural references unless the content clearly dictates otherwise.For example, an embodiment including “a compound” should be understoodto present certain aspects with one compound or two or more additionalcompounds.

In embodiments comprising an “additional” or “second” component, such asan additional or second compound, the second component as used herein ischemically different from the other components or first component. A“third” component is different from the other, first, and secondcomponents, and further enumerated or “additional” components aresimilarly different.

In embodiments of the present application, the compounds describedherein may have at least one asymmetric center. Where compounds possessmore than one asymmetric center, they may exist as diastereomers. It isto be understood that all such isomers and mixtures thereof in anyproportion are encompassed within the scope of the present application.It is to be further understood that while the stereochemistry of thecompounds may be as shown in any given compound listed herein, suchcompounds may also contain certain amounts (for example, less than 20%,suitably less than 10%, more suitably less than 5%) of compounds of thepresent application having alternate stereochemistry. It is intendedthat any optical isomers, as separated, pure or partially purifiedoptical isomers or racemic mixtures thereof are included within thescope of the present application.

The term “alkyl” as used herein, whether it is used alone or as part ofanother group, means straight or branched chain, saturated alkyl groups.The number of carbon atoms that are possible in the referenced alkylgroup are indicated by the numerical prefix “C_(n1-n2)”. For example,the term C₁₋₆alkyl means an alkyl group having 1, 2, 3, 4, 5 or 6 carbonatoms.

The term “alkylene” as used herein, whether it is used alone or as partof another group, means straight or branched chain, saturated alkylenegroup; that is a saturated carbon chain that contains substituents ontwo of its ends. The number of carbon atoms that are possible in thereferenced alkylene group are indicated by the numerical prefix“C_(n1-n2)”. For example, the term C₄₋₂₀alkylene means an alkylene grouphaving 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20carbon atoms.

The term “aryl” as used herein, whether it is used alone or as part ofanother group, refers to mono-, bi- or tricyclic groups that contain atleast one aromatic carbocycle. In an embodiment of the presentapplication, the aryl group contains 6, 9, 10 or 14 carbon atoms, suchas phenyl, naphthyl, indanyl or anthracenyl.

The term “tellurophene” as used herein refers to the a compound of theformula:

wherein the numbers are used in the naming of various substituents onthe tellurophene ring.

The term “organotellurophene” refers to a tellurophene substituted withat least one carbon-containing group.

The term “electron withdrawing group” as used herein refers to an atomor functional group that removes electron density from a conjugated πsystem, making the π system more electrophilic.

The term “unsaturated” as used herein refers to compounds or groupscomprising at least one double bond and includes compounds and groupswith a maximum number of double bonds and aromatic compounds and groups.

The terms “protective group” or “protecting group” or “PG” or the likeas used herein refer to a chemical moiety which protects or masks areactive portion of a molecule to prevent side reactions in thosereactive portions of the molecule, while manipulating or reacting adifferent portion of the molecule. After the manipulation or reaction iscomplete, the protecting group is removed under conditions that do notdegrade or decompose the remaining portions of the molecule. Theselection of a suitable protecting group can be made by a person skilledin the art. Many conventional protecting groups are known in the art,for example as described in “Protective Groups in Organic Chemistry”McOmie, J. F. W. Ed., Plenum Press, 1973, in Greene, T. W. and Wuts, P.G. M., “Protective Groups in Organic Synthesis”, John Wiley & Sons,3^(rd) Edition, 1999 and in Kocienski, P. Protecting Groups, 3rdEdition, 2003, Georg Thieme Verlag (The Americas). Examples of suitableprotecting groups include, but are not limited to t-Boc, Ac, Ts, Ms,silyl ethers such as TMS, TBDMS, TBDPS, Tf, Ns, Bn, Fmoc, benzoyl,dimethoxytrityl, methoxyethoxymethyl ether, methoxymethyl ether,pivaloyl, p-methyoxybenzyl ether, tetrahydropyranyl, trityl, ethoxyethylethers, carbobenzyloxy, benzoyl and the like.

The term “functional group” as used herein refers to a group of atoms ora single atom that will react with another group of atoms or a singleatom (so called “complementary functional group”) to form a chemicalinteraction between the two groups or atoms.

The term “complementary functional group” as used herein means afunctional group that interacts, or reacts, with another specifiedfunctional group, to form a chemical interaction. In an embodiment, thechemical interaction is a covalent bond or an ionic bond. In anotherembodiment, the chemical interaction is a covalent bond.

The term “reacts with” as used herein generally means that there is aflow of electrons or a transfer of electrostatic charge resulting in theformation of a chemical interaction.

The term “chemical interaction” as used herein refers to the formationof either a covalent of ionic bond between the reactive functionalgroups.

The term “available hydrogen atoms” as used herein refers to hydrogenatoms on a molecule that can be replaced with another group underconditions that will not degrade or decompose the parent compound. Suchconditions include the use of protecting groups to protect sensitivefunctional groups in the molecule while the hydrogen atom is beingreplaced.

The term “compound(s) of the application” or “compound(s) of the presentapplication” and the like as used herein includes organotellurophenecompounds comprising a tellurophene moiety, a linker and a reactivefunctional group wherein the reactive functional group is capable ofbeing functionalized with a biosensor, a biologically active materialand/or a polymeric backbone, organotellurophene compounds comprising atellurophene moiety, a linker and a biosensor, a biologically activematerial and/or a polymeric backbone and particularly compounds offormula (I), (II) and (IIa), and pharmaceutically acceptable saltsand/or solvates thereof as defined herein.

An acid addition salt means any organic or inorganic salt of any basiccompound. Basic compounds that form an acid addition salt include, forexample, compounds comprising an amine group. Illustrative inorganicacids which form suitable salts include hydrochloric,hydrotrifluoroacetic, hydrobromic, sulfuric and phosphoric acids, aswell as metal salts such as sodium monohydrogen orthophosphate andpotassium hydrogen sulfate. Illustrative organic acids that formsuitable salts include mono-, di-, and tricarboxylic acids such asglycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic,tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic andsalicylic acids, as well as sulfonic acids such as p-toluene sulfonicand methanesulfonic acids. Either the mono or di-acid salts can beformed, and such salts may exist in either a hydrated, solvated orsubstantially anhydrous form. In general, acid addition salts are moresoluble in water and various hydrophilic organic solvents, and generallydemonstrate higher melting points in comparison to their free baseforms. The selection of the appropriate salt will be known to oneskilled in the art.

A base addition salt means any organic or inorganic base addition saltof any acidic compound. Acidic compounds that form a base addition saltinclude, for example, compounds comprising a carboxylic acid group.Illustrative inorganic bases which form suitable salts include lithium,sodium, potassium, calcium, magnesium or barium hydroxide. Illustrativeorganic bases which form suitable salts include aliphatic, alicyclic oraromatic organic amines such as methylamine, trimethylamine andpicoline, alkylammonias or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The term “solvate” as used herein means a compound of the formula (I) orformula II or a pharmaceutically acceptable salt of a compound of theformula (I) or formula (II), wherein molecules of a suitable solvent areincorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”. The formation ofsolvates of the compounds of the application will vary depending on thecompound and the solvate. In general, solvates are formed by dissolvingthe compound in the appropriate solvent and isolating the solvate bycooling or using an antisolvent. The solvate is typically dried orazeotroped under ambient conditions.

As used herein, the term “effective amount” means an amount effective,and for periods of time necessary, to achieve a desired result.

The term “polymeric backbone” as used herein refers to the main chain ofa suitable polymer comprising a series of covalently bonded atoms thattogether create the continuous chain (straight or branched) of thepolymeric molecule. The polymer is any suitable polymer or copolymercomprising at least one compound of formula (IIa) covalently linkedthereto. In some embodiments, the polymeric backbone comprisesfunctional atoms that increase water solubility, for example,polyethyleneglycol units, and/or, attached functional groups thatincrease water solubility, for example, zwitter ionic groups. In someembodiments, the polymeric backbone is coupled to and/or furthercomprises one or more biosensors, biologically active materials, masstags and/or a supporting structure of a mass tag.

The definitions and embodiments described in particular sections areintended to be applicable to other embodiments herein described forwhich they are suitable as would be understood by a person skilled inthe art. For example, in the following passages, different aspects aredefined in more detail. Each aspect so defined may be combined with anyother aspect or aspects unless clearly indicated to the contrary. Inparticular, any feature indicated as being preferred or advantageous maybe combined with any other feature or features indicated as beingpreferred or advantageous.

II. Compounds, Compositions and Kits

Functionalized organotellurophene compounds as probes for mass cytometry(MC) have been prepared as described in the present application. Theorganotellurophene compounds were characterized by nuclear magneticresonance spectroscopy and their stability monitored throughultraviolet-visible spectroscopy. The metabolic toxicity and theirsubsequent potential as MC probes have also been assessed in the studiesof the present application.

Accordingly, the present application includes an organotellurophenecompound, comprising a tellurophene moiety, a linker and a reactivefunctional group wherein the reactive functional group is capable ofbeing functionalized with a biosensor, a biologically active material ora polymeric backbone. In a further embodiment, the present applicationincludes an organotellurophene compound, comprising a tellurophenemoiety, a linker and a biosensor, a biologically active material and/ora polymeric backbone.

In an embodiment, the organotellurophene compounds of the applicationcomprise a tellurophene optionally functionalized at the 5-position witha bulky group and/or an electron withdrawing group and at the 2-positionwith a linker group that is attached to a reactive functional group X.In another embodiment, the organotellurophene compounds comprise of atellurophene optionally functionalized at the 5-position with a bulkygroup and/or an electron withdrawing group and at the 2-position with alinker group that is attached to a biosensor, a biologically activematerial and/or a polymeric backbone. It is an embodiment that thesubstituent at the 5-position helps to stabilize the organotellurophenecompound, for example by inhibiting oxidation of the Te atom.

In another embodiment, the present application includes anorganotellurophene compound of formula (I):

wherein A is a naturally occurring isotope of Te;R¹ is selected from H, unsubstituted or substituted C₁-C₂₀alkyl,unsubstituted or substituted C₃-C₂₀cycloalkyl, unsubstituted orsubstituted aryl and an electron withdrawing group;L is C₁₋₃₀alkylene, unsubstituted or substituted with one or moresubstituents and/or optionally interrupted with one or moreheteromoieties independently selected from O, S, NR⁷, and/or optionallyinterrupted with one or more of C(O) and C(S);R⁷ is independently selected from H, PG and C₁₋₆alkyl;X is a reactive functional group selected from halo, OH, OTs, OMs,C(O)H, C(O)OR⁸, C(O)NR⁹R¹⁰, O—C(O)—OR¹¹, O—C(O)—NR¹², C(O)ONR¹³R¹⁴,C(O)R¹⁵, C(O)SR¹⁶ and NR¹⁷R¹⁸;R⁸ is selected from H, C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, the latterthree groups being unsubstituted or substituted with one or more of haloand NO₂;R⁹ and R¹⁰ are independently selected from H, C₁₋₆alkyl, aryl andC₁₋₆alkylenearyl, the latter three groups being unsubstituted orsubstituted with one or more of halo and NO₂, orR⁹ and R¹⁰, together with the N atom to which they are bonded, form a 4to 12 membered monocyclic or bicyclic, saturated or unsaturated ringunsubstituted or substituted with one or more ═O, ═S, halo andC₁₋₆alkyl;R¹¹ is selected from C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, the latterthree groups being unsubstituted or substituted with one or more of haloand NO₂;R¹² is selected from C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, the latterthree groups being unsubstituted or substituted with one or more of haloand NO₂;R¹³ and R¹⁴ are independently selected from H, C₁₋₆alkyl, aryl andC₁₋₆alkylenearyl, the latter three groups being unsubstituted orsubstituted with one or more of halo and NO₂, orR¹³ and R¹⁴, together with the N atom to which they are bonded, form a 4to 12 membered monocyclic or bicyclic, saturated or unsaturated ringunsubstituted or substituted with one or more ═O, ═S, halo andC₁₋₆alkyl;R¹⁵ is halo;R¹⁶ is selected from C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, the latterthree groups being unsubstituted or substituted with one or more of haloand NO₂:R¹⁷ and R¹⁸ are independently selected from H, C(O)C₁₋₆ alkyl,C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, the latter three groups beingunsubstituted or substituted with one or more of halo and NO₂, orR¹⁶ and R¹⁷, together with the N atom to which they are bonded, form a 4to 12 membered monocyclic or bicyclic, saturated or unsaturated ringunsubstituted or substituted with one or more ═O, ═S, halo andC₁₋₆alkyl; andone or more available hydrogens are optionally replaced with D;or a salt and/or solvate thereof;with proviso that when X is C(O)OR¹⁹ and R¹⁹ is H or C₁₋₆alkyl, L is notC₁₋₈alkylene; andwhen X is OH, L is not C₁₋₂alkylene.

In an embodiment, R¹ in the compounds of formula (I) is an electronwithdrawing group selected from C(O)R², C(R³)₃, C≡N, and NO₂, wherein R²is selected from H and C₁₋₆alkyl and R³ is halo.

In an embodiment, the substituents on R¹ in the compounds of formula (I)are independently selected from one or more of halo, C₁₋₆ alkyl andC₁₋₆alkoxy.

It is a further embodiment that R¹ in the compounds of formula (I) is H,unsubstituted or substituted C₁-C₁₀alkyl, unsubstituted or substitutedC₃-C₁₀cycloalkyl, unsubstituted or substituted phenyl and an electronwithdrawing group selected from C(O)R² and C(R³)₃. In anotherembodiment, the substituents on R¹ are independently selected from oneor more of halo and C₁₋₃alkyl, R² is selected from H and C₁₋₆alkyl; andR³ is F, Cl, Br, and I. In a further embodiment, R¹ is selected from Hand C(R³)₃ wherein R³ is F. It is an embodiment that R¹ is H.

In an embodiment, the substituents on L in the compounds of formula (II)are independently selected from halo, C₁₋₆alkyl, C₁₋₆alkoxy, C(O)R⁴ andNR⁵R⁶, wherein R⁴ is selected from H and C₁₋₆alkyl; and R⁵ and R⁶ areindependently selected from H, PG, C(O)C₁₋₂₀alkyl and C(O)OC₁₋₂₀ alkyl.

In an embodiment, L in the compounds of formula (I) is a C₁₋₂₅alkylene,unsubstituted or substituted with one or more substituents independentlyselected from C₁₋₃alkyl, C(O)R⁴ and NR⁵R⁶, and/or optionally interruptedwith one or more heteromoieties independently selected from O and NR⁷,and/or optionally interrupted with C(O), R⁴ is selected from H andC₁₋₂alkyl, R⁵ and R⁶ are independently selected from H, PG,C(O)C₁₋₆alkyl and C(O)OC₁₋₆alkyl, and R⁷ is independently selected fromH and PG.

In another embodiment, L in the compounds of formula (I) is aC₁₋₂₅alkylene, unsubstituted or substituted with one or moresubstituents independently selected from NR⁵R⁶, and/or optionallyinterrupted with one or more heteromoieties independently selected fromO and NR⁷, and/or optionally interrupted with C(O); R⁵ and R⁶ areindependently selected from H, PG, and C(O)OC₁₋₄alkyl; and R⁷ is H.

In an embodiment, X in the compounds of formula (I) is a reactivefunctional group selected from Cl, Br, I, OH, C(O)OR⁸, C(O)NR⁹R¹⁰,O—C(O)—OR¹¹, C(O)ONR¹³R¹⁴, C(O)R¹⁵ and NR¹⁵R¹⁸; R⁸ is selected from Hand C₁₋₂alkyl; R⁹ and R¹⁰ are independently selected from H, C₁₋₆alkyl,aryl and C₁₋₆alkylenearyl, wherein the latter three groups areunsubstituted or substituted with one or more of halo and NO₂; R¹¹ is aphenyl, unsubstituted or substituted with one or more of F, Cl, Br, Iand NO₂, R¹³ and R¹⁴ are independently selected from H and C₁₋₂alkyl orR¹³ and R¹⁴, together with the N atom to which they are bonded, form a 4to 10 membered monocyclic or bicyclic ring unsubstituted or substitutedwith one or more ═O, and ═S; R¹⁵ is Cl or Br; and R¹⁷ and R¹⁸ areindependently selected from H, C(O)C₁₋₃alkyl or R¹⁷ and R¹⁸, togetherwith the N atom to which they are bonded, form a 4 to 12 memberedmonocyclic or bicyclic ring unsubstituted or substituted with one ormore ═O and ═S.

In another embodiment, X in the compounds of formula (I) is a reactivefunctional group selected from Cl, OH, C(O)OR⁸, C(O)NR⁹R¹⁰, O—C(O)OR¹¹,C(O)ONR¹³R¹⁴ and NR¹⁷R¹⁸; wherein R⁸ is H; R⁹ and R¹⁰ are independentlyselected from H, C₁₋₆alkyl and C₁₋₆alkylenearyl, wherein the latterthree groups are unsubstituted or substituted with one or more of haloand NO₂; R¹¹ is a phenyl substituted with NO₂, R¹³ and R¹⁴ together withthe N atom to which they are bonded, form a 4 to 6 membered monocyclicring substituted with ═O; and R¹⁷ and R¹⁸ are independently selectedfrom H, C(O)C₁₋₂alkyl or R¹⁷ and R¹⁸, together with the N atom to whichthey are bonded, form a 4 to 10 membered bicyclic ring substituted with═O.

In an embodiment, the compound of formula (I) is selected from:

In an embodiment, the present application also includes anorganotellurophene compound of formula (II):

wherein A is a naturally occurring isotope of Te;R¹ is selected from H, unsubstituted or substituted C₁-C₂₀alkyl,unsubstituted or substituted C₃-C₂₀cycloalkyl, unsubstituted orsubstituted aryl and an electron withdrawing group;L is C₁₋₂₀alkylene, unsubstituted or substituted with one or moresubstituents and/or optionally interrupted with one or moreheteromoieties independently selected from O, S, NR⁷, and/or optionallyinterrupted with one or more of C(O) and C(S);R⁷ is independently selected from H, PG and C₁₋₆alkyl. andZ is a biosensor, biologically active material, or polymeric backbone;andor a salt and/or solvate thereof.

In an embodiment, R¹ in the compounds of formula (II) is an electronwithdrawing group selected from C(O)R², C(R³)₃, C≡N, and NO₂, wherein R²is selected from H and C₁₋₆alkyl and R³ is halo.

In an embodiment, the substituents on R¹ in the compounds of formula(II) are independently selected from one or more of halo, C₁₋₆ alkyl andC₁₋₆alkoxy.

In another embodiment, R¹ in the compounds of formula (II) is selectedfrom H, unsubstituted or substituted C₁-C₁₀alkyl, unsubstituted orsubstituted C₃-C₁₀cycloalkyl, unsubstituted or substituted phenyl and anelectron withdrawing group selected from C(O)R² and C(R³)₃; thesubstituents on R¹ are independently selected from one or more of haloand C₁₋₃alkyl; R² is selected from H and C₁₋₆ alkyl; and R³ is F, Cl,Br, and I.

In a further embodiment, R¹ in the compounds of formula (II) is selectedfrom H and C(R³)₃ wherein R³ is F.

In yet a further embodiment, R¹ in the compounds of formula (II) is H.

In an embodiment, the substituents on L in the compounds of formula (II)are independently selected from halo, C₁₋₆alkyl, C₁₋₆alkoxy, C(O)R⁴ andNR⁵R⁶, wherein R⁴ is selected from H and C₁₋₆alkyl; and R⁵ and R⁶ areindependently selected from H, PG, C(O)C₁₋₂₀alkyl and C(O)OC₁₋₂₀ alkyl.

In an embodiment, L in the compounds of formula (II) is a C₁₋₂₅alkylene,unsubstituted or substituted with one or more substituents independentlyselected from C₁₋₃alkyl, C(O)R⁴ and NR⁵R⁶, and/or optionally interruptedwith one or more heteromoieties independently selected from O and NR⁷,and/or optionally interrupted with C(O); R⁴ is selected from H andC₁₋₂alkyl; R⁵ and R⁶ are independently selected from H, PG,C(O)C₁₋₆alkyl and C(O)OC₁₋₆alkyl; and R⁷ is independently selected fromH and PG.

In another embodiment, L in the compounds of formula (II) is aC₁₋₂₅alkylene, unsubstituted or substituted with one or moresubstituents independently selected from NR⁵R⁶, and/or optionallyinterrupted with one or more heteromoieties independently selected fromO and NR⁷, and/or optionally interrupted with C(O); R⁵ and R⁶ areindependently selected from H, PG, and C(O)OC₁₋₄alkyl; and R⁷ is H.

In an embodiment, Z in the compounds of formula (II) is a biosensor.

In an embodiment, biosensor is an oxidoreductase substrate, such as axanthine oxidase substrate or a P450 substrate. As described herein,xanthine oxidase catalyzes the reduction the of 2-nitroimidazolecomponent of Telox and Telox2.

In another embodiment, the biosensor further comprises a mass tag or asupporting structure of a mass tag, wherein the mass tag or supportingstructure is optionally directly attached to the biosensor or attachedthrough a linker, such as a linker L as defined herein.

In another embodiment, the biosensor is 2-nitroimidazole.

In a further embodiment, the compound of formula (II) is selected from:

As demonstrated herein, these compounds could be used to label cellsunder hypoxic conditions. Such compounds when incubated with cells underlow oxygen conditions undergo an enzyme catalyzed reduction of the2-nitroimidazole functionality to produce the electrophilicprotein-labelling nitrenium ion which forms adducts, allowing labellingof hypoxic cells.

In an embodiment, a series of these compounds, each comprising one ormore different isotopes of Te can be used, for example, to profiledifferent test variables in a single mass cytometry.

A variety of enzyme substrates can be used as biosensors including forexample substrates for oxidoreductases, glycosyl hydrolases, lipases,phosphatases, kinases and proteases Active site specific reactivecompounds specific for proteases, for, example comprising anelectrophilic group which covalently binds to the catalyticnucleophiles, (e.g. Ser, Cys or Thr in serine, cysteine and threonineproteases respectively) located at the active site of the enzymes.

Other biosensors include for example protease substrates that produce areactive product or are irreversible inhibitors that selectively labelactive enzymes. In an embodiment, the protease is a cysteine proteasesubstrate.

Other biosensors include for example glycosyl hydrolase substrates. Inan embodiment the glycosyl hydrolase substrate is a B-galactosidasesubstrate including for example compound 24 described herein. ThisB-galactosidase substrate upon substrate cleavage produces a quinonemethide tellurophene labelled compound that reacts with cell componentsthereby labelling the cell.

In one embodiment, the reactive product is a quinone methide.

In an embodiment, the biosensor comprises a membrane targeting moietysuch as a fatty acid. The fatty acid can for example comprise analiphatic tail with at least 4 carbons, at least 6 carbons or any numberof carbons between 4 and 22 carbons

In an embodiment, the biosensor is a cathepsin protease substrate, suchas a cathepsin S protease substrate. In an embodiment, the tellurophenetagged biosensor comprises

The compound is a cathepsin S peptide substrate and is membraneassociated. In an embodiment, the substrate is labelled with two masstags, optionally two organotellurophene moieties each comprising adifferent isotope of Te. In such an embodiment, cellular cathepsin Sactivity results in cleavage of the substrate and release of one of themass tags. A ratio of the two mass tags can be calculated and isindicative of cathepsin activity.

Yet other biosensors include for example phosphatase substrates such asalkaline phosphatase substrates, as well as ATPase active site-specificreactive compounds, GTPase active site-specific reactive compounds andkinase active site-specific reactive compounds

In another embodiment, Z in the compounds of formula (II) is abiologically active material.

In a further embodiment, the biologically active material is selectedfrom a cell, virus, subcellular particle, polypeptide, nucleic acid,peptidic nucleic acid, oligosaccharide, polysaccharide,lipopolysaccharide, cellular metabolite, hapten, hormone,pharmacologically active substance, alkaloid, steroid, vitamin, aminoacid and sugar.

In yet a further embodiment, the biologically active material isselected from a polypeptide, oligosaccharide, polysaccharide,lipopolysaccharide, sugar, cellular metabolite, pharmacologically activesubstance and amino acid.

In yet another embodiment, the biologically active material is selectedfrom a sugar, pharmacologically active substance and amino acid.

In an embodiment, the amino acid is lysine, phenylalanine, tyrosine oror tryptophan.

In another embodiment, the biologically active material is an affinityreagent selected from an antibody or binding fragment thereof, aptamer,avidin reagent, nucleic acid or lectin.

In another embodiment, the biologically active material furthercomprises a mass tag or a supporting structure of a mass tag, whereinthe mass tag or supporting structure is optionally directly attached tothe biosensor or attached through a linker, such as a linker L asdefined herein.

In yet a further embodiment, the compound of formula (II) is selectedfrom:

In an embodiment, Z in the compounds of formula (II) is a monomeric unitof a polymeric backbone and the compound comprises at least one offormula (IIa):

wherein n is an integer representing the number of repeating monomericunits of formula (IIa).

In another embodiment, the polymeric backbone further comprises monomerscontaining negative charges and/or side chains that improve watersolubility.

In a further embodiment, the monomers that improve water solubilitycomprise of polyethyleneglycol units and/or zwitter ions.

In another embodiment, the polymeric backbone further comprises one ormore biosensors, and/or biologically active materials, optionallydirectly attached or attached through a linker, such as linker L asdefined herein. In another embodiment, polymeric backbone furthercomprises a mass tag or a supporting structure of a mass tag, whereinthe mass tag or supporting structure is optionally directly attached tothe biosensor or attached through a linker, such as a linker L asdefined herein.

In an embodiment, the polymer backbone is one described in Lou et al,Angew. Chem Int Ed 2007 [42], or Majonis et al, Anal Chem 2010 [43],each hereby incorporated by reference.

In yet a further embodiment, the compound of formula (IIa) is selectedfrom:

wherein Q is O or NH.

In an embodiment, a compound of formula (I), a compound of formula (II)or a compound of formula (IIa) comprise a tellurium isotope selectedfrom ¹²⁰Te, ¹²²Te, ¹²³Te, ¹²⁴Te, ¹²⁵Te, ¹²⁶Te, ¹²⁸Te and ¹³⁰Te.

The compounds described herein can be attached to a solid support suchas a bead, slide, synthetic membrane, plate, tube or column. Forexample, the bead can be an agarose bead or a silica bead The solidsupport can comprise one or more different compounds and/or a distincttellurium mass and/or distinct tellurium isotope such that it isdistinguishable from other types of solid supports by tellurium massanalysis.

The compound of formula (I) are prepared using methods known in the artfrom materials that are either commercially available or are alsoprepared using methods known in the art. For example, the compounds offormula (I) are prepared by combining a compound of formula (III), or aprotected form thereof:

wherein R¹, L and X are as defined above, with an aqueous suspension ofone or more naturally occurring isotopes of Te⁰ and a basic Rongalitesolution under conditions to provide the compound of formula (I). Thecompounds of formula (II) are prepared from the compounds of formula (I)by reacting a suitable precursor to the biosensor, biologically activematerial, or polymeric backbone, the suitable precursor comprising acomplementary functional group to X.

The conversion of a compound of formula (I) to a compound of formula(II), is performed using methods known in the art, for example usingnucleophilic addition conditions and activated acid substitutionconditions. The polymeric backbone and attachments of biosensorsbiologically active materials, mass tags and/or mass tag supportingstructures thereto are made using methods known in the art, for example,as described in U.S. Pat. No. 9,012,239, herein incorporated byreference.

The reaction conditions will depend, for example, on the identity of thereactive functional group and the biosensor, biologically activematerial, mass tag, supporting structure in a mass tag and/or polymericbackbone and may involve one or more presteps. For example thebiologically active material may be pre-treated to activate groups thatcan react with the organotellurophene tag functional group and/or theorganotellurophene tag may be pre-treated to activate groups that canreact with the biologically active material. Further, the reactions mayneed to be modified to include the use of protecting groups.

Purifying the mass tagged biosensor, biologically active material and/orpolymer can comprise removing unreacted starting materials and cancomprise one or more steps of filtering, column purification,centrifuging and/or washing and recovering the mass tagged biosensor,biologically active material or polymer.

In an embodiment, the present application also includes compositionscomprising one or more compounds selected from a compound of formula (I)and a compound of formula (II), and salts and/or solvates thereof. In anembodiment, the composition further comprises a carrier. Examples ofcarriers include, but are not limited to, solvents, adjuvants andexcipients. In a further embodiment the composition further comprisesother components, for example, for the stability, of the composition,such as antioxidants and/or antimicrobial agents. In an embodiment, thecomposition comprising one or more compounds of formula (II), and/orsalts and/or solvates thereof, is compatible with biological systems,including cells. In an embodiment, “compatible with” means non-toxic to,or at least having a toxicity that is below acceptable levels.

In an embodiment, the compositions of the application comprise aplurality of compounds of formula (I) and/or (II) each having adifferent tellurium isotope.

In an embodiment, the compositions of the application comprise aplurality of compounds of formula (II), each having a differentbiosensor, a different biologically active material (optionally adifferent antibody) and/or a different polymer.

In an embodiment, the compositions of the application comprise aneffective amount of one or more compounds selected from a compound offormula (I) and a compound of formula (II), and salts and/or solvatesthereof.

In an embodiment, the compound or composition of the application iscomprised in a vial. For example, the vial is a light blocking vial forlight sensitive compounds or compositions. In an embodiment, thecompounds or compositions are stored in the vial under inert atmosphericconditions, particularly for example for oxygen reactive compounds.

In an embodiment, the application includes a kit comprising a compound,composition or vial described herein and instructions or reagents forreconstituting and/or using the compound or composition in, for example,a mass detection assay. For example the kit can comprise an alkalinephosphatase substrate tagged with a tellurophene compound of formula(I). In an embodiment, the instructions are for mass tagging abiosensor, biologically active material or a polymer backbone with acompound of formula (I) or performing a mass detection assay with themass tagged biosensor or biologically active material. In an embodiment,the mass detection assay is a mass cytometry assay.

In an embodiment, the kit is a multiplex kit and comprises at least 2,3, 4, 5, 6, 7 or 8 compounds, each compound comprising a differenttellurium isotope, different combinations of tellurium isotopes suchthat the compounds have a distinct tellurium mass and/or a differentbiosensor, a different biologically active compound and/or polymericbackbone. The kit can comprise a series of compounds which are the samecompound other than the tellurium isotope or they can be differentcompounds comprising different tellurium epitopes. Examples include aplurality of compounds of formula (I), each compound having the samestructure and comprising a different tellurium isotope. Alternatively,the compounds can be compounds of formula (II), optionally wherein thebiologically active material is for example an affinity reagent, such asan antibody specific for a particular antigen, with each compoundcomprising a different tellurium isotope.

The compounds, compositions, and kits described herein includecomponents and/or can be packaged for particular assays.

In an embodiment, the kit comprises a standard such as an internalstandard for example a calibration bead for use in mass cytometryapplications.

III. Methods and Uses

One aspect described herein includes a method of mass tagging abiosensor, biologically active material or polymer backbone or the useof an organotellurophene tag (e.g. a compound of formula (I)) forpreparing a mass tagged biosensor, a mass tagged biologically activematerial or a mass tagged polymer.

In an embodiment, the method comprises contacting an organotellurophenetag, comprising a linker and a reactive functional group with abiosensor, biologically active material or polymeric backbone undersuitable reaction conditions; and purifying the mass tagged biosensor ormass tagged biologically active material.

In an embodiment, the organotellurophene tag is a compound of formula(I).

Different biosensors, biologically active materials, and polymericbackbones are described herein and can be employed in the methods anduses described herein. Synthetic schemes for a number of compounds offormula (II) are provided below. Accordingly, in an embodiment themethod of mass tagging produces a compound of formula (II). In anembodiment the method employs a synthetic scheme described herein.

In an embodiment, the method contacting step comprises contacting abiologically active material selected from a cell, virus, subcellularparticle, polypeptide, nucleic acid, peptidic nucleic acid,oligosaccharide, polysaccharide lipopolysaccharide, cellular metabolite,hapten, hormone, pharmacologically active substance, alkaloid, steroid,vitamin, amino acid and sugar with the tellurophene tag under suitableconditions.

In another embodiment, the biologically active material is selected froman affinity reagent selected from an antibody or binding fragmentthereof, aptamer, avidin reagent, nucleic acid or lectin. Thebiolologically active material can be tagged to the tellurophene tagthrough for example a thiol of a cysteine residue or a thiol engineeredinto the biologically active material. Reaction of the thiol with athiol selective reagent, for example, a maleimide will give the desiredconstruct. Alternatively free amines on the biologically active materialcan be acylated by the tellurophene tag.

The compounds described herein can be used in several assays includingcytometry assays that can use fluorescent markers. For example,tellurophene mass tagged compounds as described herein can be coupled toaffinity reagents such as antibodies, oligonucleotides, lectins,apatamers and the like and used for detecting a target analyte,optionally in or on a cell.

In particular, the compounds can be used for multiplex labelling ofcells, viruses, subcellular particles, polypeptides, nucleic acids andthe like. For example, mass tagged biologically active materials, suchas mass tagged affinity reagent such as antibodies can be prepared asdescribed for a number of target analytes. In an example, each masstagged affinity reagent is directed to a different analyte and comprisesa distinct tellurium mass or is used in combination with othernon-tellurium mass tagged molecules to expand the number of parametersthat can be assayed. Cells can be cultured under normal conditions,labelled with a desired combination of mass tagged affinity reagents inone reaction mixture to assay multiple parameters of a single cellpopulation. Alternatively, cells can be labelled with affinity reagentsto one or more target analytes in different reaction mixtures to assayone or more test parameters, wherein each reaction mixture is a cellpopulation treated under a different test parameter. The cells can bewashed, collected, fixed, optionally stained with one or moreintercalators such as a Rhodium based nucleic acid intercalator (e.gMaxPar® Intercalotor-Rh, Fluidigm) to distinguish dead from live cellsand/or singly nucleated cells from other cells and analysed by masscytometry, for example as described for Telox/Telox2 hypoxia examplesdescribed herein.

Accordingly another aspect includes a method of detecting or quantifyinga target activity or target analyte comprising the steps of:

-   -   providing a cell or cell population;    -   providing a tellurophene tagged biosensor or biologically active        material optionally a compound of formula (II), wherein the        biosensor is a substrate for the target activity and/or the        biologically active material specifically binds the target        analyte;    -   mixing the cell or cell population with the tellurophene tagged        biosensor or biologically active material; and    -   detecting tellurium labelling and/or quantitating the amount of        tellurium labelling of the cell or cell population.

The target analyte can for example be a cell surface or intracellularentity in a cell of the cell population. Similarly, the activity can bea cell surface or intracellular enzymatic activity. As the tags can becompact, the tags can be used to label intracellular constituents aswell as extracellular antigens. Assaying the tellurium labelling of thecell population indicates whether the target analyte or activity ispresent and/or the amount of analyte or activity.

Assays for detecting and/or quantitating the tellurium labelling of thetarget analyte and/or cell population include mass based methods whichcan monitor the distinct tellurium mass or distinct tellurium isotopesuch as a mass cytometry assay.

As described herein, detecting and/or quantitating the telluriumlabelling using mass cytometry involves vaporizing cells and analyzingsaid cells by time of flight mass spectrometry.

Mass cytometry, in addition to enabling single cell analysis can includemass cytometry imaging methods for example as described in (Giesen et al2014, incorporated herein by reference). In such methods, a tissue orcell population is labelled in vitro with mass tagged biosensors and/orbiologically active materials, the tissue or cell population issubjected to laser ablation coupled to mass cytometry and the telluriumsignal processed to provide an image showing single cell segmentation.Different tissue preparations can be used including for example formalinfixed and fresh tissue.

In other embodiments, the substrate may produce an insoluble telluriumcontaining compound that precipitates locally. The presence of theprecipitate is imaged, and can provide an indication of enzymelocalization in a cell or tissue.

The detecting and/or quantitating tellurium labelling can also employ anenzyme linked assay. In enzyme linked assays, the enzyme substrate,optionally an alkaline phosphatase substrate, is tagged with atellurophene tag. The substrate upon cleavage produces a reactiveproduct such as a quinone methide. In the presence of enzyme, thereactive tellurium containing product would be formed and wouldcovalently label the enzyme or other local biomolecules. Telluriumpresence can be measured and is indicative of the presence and/or amountof enzyme or enzyme activity.

Different tellurophene tags each comprising distinct mass can be used toanalyse a number of parameters in parallel.

In an embodiment, a tellurophene tagged biologically active material isprovided. In an embodiment, the target analyte is a polypeptide and thebiosensor biologically active material is a polypeptide affinity reagentoptionally an antibody, aptamer avidin reagent such as streptavidin, ordeglycosylated avidin.

Nucleic acid probes can also be prepared comprising a tellurophene tag.Accordingly in an embodiment, the target analyte is a nucleic acid andthe biologically active material is a polynucleotide probe.

In an embodiment, a tellurophene tagged biosensor is provided.

As demonstrated herein, in addition to looking at static biomarkers,tellurophene tagged biosensors such as the ones described herein can beused advantageously to probe cellular enzymatic and/or metabolicactivity. In an embodiment, the tellurium labelling is indicative of thepresence or amount target activity

For example, compounds comprising the hypoxia sensitive biosensor2-nitroimidazole are described. These compounds as demonstrated hereincan be used for detecting or labelling oxygen deprived cells. Telox andTelox2 as well as other 2-nitroimidizole comprising compounds areenzymatically processed and form reactive intermediates under low oxygenconditions. Said reactive intermediates form adducts thereby labelingthe cells. Assaying the cells for tellurium isotopes identifies cellsthat exposed to low oxygen conditions.

Accordingly in an embodiment, the activity is oxidoreductase activity.In an embodiment, the method is for detecting and/or labelling oxygendeprived cells, the method comprising: incubating a population of cellswith the compound of formula (II) wherein Z is a biosensor comprising2-nitroimidazole, detecting and/or quantitating the amount of telluriumlabelling in the cell population, wherein tellurium labelling isindicative of oxygen deprivation.

The population of cells can for example be in a tissue sample or can besubjected to different test conditions to assess whether hypoxia isinduced. In an embodiment, the tellurium labelling is measured by masscytometry.

In an embodiment, the method further comprises quantifying the number ofoxygen deprived cells in the population.

In an embodiment, the method comprises one or more of the steps in FIG.4 or described herein. In an embodiment, the quantitating comprisescomparing to a control.

Other enzyme activities can also be measured using organotellurophenemass tagged compounds. Proteases comprise an active site that isavailable to substrates when the protease is active. For example, acathepsin S substrate is described that can be used to measure cathepsinS activity, the cathepsin S substrate being

In the presence of active cathepsin, cleavage of the substrate resultsin production of a tellurium containing peptide (tellurium isotope 1)which is localized to the membrane. A second isotope (or other metaltag, optionally a lanthanide) can be bound to the DTPA portion of themolecule, and is released as a soluble entity after protease cleavage.The ratio of tellurium isotope 1 to the other isotope (or other metaltag) can be used to quantify the level of cathepsin activity.

Accordingly, in an embodiment, the activity is protease activity. Inanother embodiment, the activity is cathepsin S activity.

In another embodiment, the activity is glycosylhydrolase activity.Described herein is a B-galactosidase tellurophene tagged substrate(compound 24). Upon cleavage by B-galactosidase a quinone methide isproduced. Cells that comprise active B-galactosidase are labelled andcan be detected by for example mass cytometry methods.

B-galactosidase or LacZ is used in a number of applications. Differenttellurophene isotopes alone or in combination with other metal tags canfor example be used to assess the B-galactosidase activity of a seriesof clones.

In another embodiment, the activity is a phosphatase activity, kinaseactivity or lipase activity. In an embodiment, the phosphatase activityis alkaline phosphatase.

In an embodiment, the biosensor is selected from 2-nitroimidazole,oxidoreductatse substrate, protease substrate, phosphatase substrate,kinase substrate, glycosyl hydrolase substrate or lipase substrate.

Amino acids such as lysine, phenylalanine, tyrosine and tryptophan canalso be mass tagged. In addition, nucleotides, sugars can also be masstagged. Such organotellurophene tagged reagents can be used formetabolic labelling. In an embodiment, the method comprises incubating apopulation of cells in media wherein a natural molecular building blockis replaced with a mass tagged analog, the incubation being underconditions and for sufficient time for target analyte biomoleculesynthesis, for example to allow incorporation of the mass tagged analoginto the biomolecule target analyte, detecting and/or measuring thetellurium labelling of the cell population and/or target analyte by forexample mass cytometry.

In some embodiments, a plurality of target analytes and/or targetactivities are detected and/or quantified and the method comprisesproviding a plurality of tellurophene tagged biosensors and/orbiologically active materials, optionally a plurality of compounds offormula (II), wherein each compound comprises a different biosensor ordifferent biologically active material, optionally an affinity reagentand a different tellurium isotope, thereby allowing multiplexing.

The above disclosure generally describes the present application. A morecomplete understanding can be obtained by reference to the followingspecific examples. These examples are described solely for the purposeof illustration and are not intended to limit the scope of theapplication. Changes in form and substitution of equivalents arecontemplated as circumstances might suggest or render expedient.Although specific terms have been employed herein, such terms areintended in a descriptive sense and not for purposes of limitation.

The following non-limiting examples are illustrative of the presentdisclosure:

Examples Abbreviations and Definitions

CDCl₃=deuterated chloroform: DART MS=direct analysis in real time massspectrometry; DCC=N,N′-dicyclohexylcarbodiimide; DCU=dicyclourea;DCM=dichloro methane; DME=dimethyl ethane; DMSO=dimethyl sulphoxide;ESI=electrospray ionization; EtOAc=ethyl acetate; FBS=fetal bovineserum; HPLC=high performance liquid chromatography; LC-MS=liquidchromatography mass spectrometry; MC=mass cytometry;NHS=N-hydroxysuccinimide; NMR=nuclear magnetic resonance; O.N=overnight; PBS: phosphate buffered saline; p-NP=para-nitrophenol; ppm=partsper million; RPMI=Roswell Park Memorial Institute; THF=tetrahydrofuran;TLC=thin layer chromatography; WST-1=water soluble tetrazolium salt 1

Methods Instrumentation:

Rotary evaporation was performed using a Heidolph rotary evaporator. AllNMR spectra were recorded at 25° C. on one of the followingspectrometers: Agilent DD2 600 MHz (with OneNMR H/F{X} probe), AgilentDD2 500 MHz (with Xsens cold probe), or a Varian 400 MHz (with AutoXprobe). One-dimensional proton and carbon chemical shifts are reportedin parts per million and referenced to residual proton signals of NMRsolvents (CD3OD; δ 3.31 ppm, CDCl3; 7.26 ppm). All coupling constantsare reported in hertz (Hz) and protons multiplicities are described aseither s=singlet, d=doublet, dd=doublet of doublets, t=triplet,q=quartet, p=pentet, or m=multiplet. NMR data is reported in thefollowing order/format; chemical shift (multiplicity, integration,coupling constant, assignment). High-resolution mass spectra wererecorded using a JEOL AccuTOF mass spectrometer with a direct analysisin real time (DART) ionization source. ICP-MS data was obtained using aPerkinElmer ELAN-9000 spectrometer. Mass cytometry data was obtainedusing a second-generation CyTOF (DVS Sciences/Fluidigm). UV-Visspectroscopy data was recorded using an Agilent ultraviolet-visiblephotospectrometer (model #8453).

High resolution mass spectrometry was obtained by one of the following:JEOL AccuTOF model JMS-T1000LC mass spectrometer equipped with DART ionsource or Agilent 6538 Q-TOF mass spectrometer equipped with Agilent1200 HPLC and an ESI ion source.

Reagents and General Conditions:

Solvents were removed under vacuum at approximately 40° C. All reactionswere performed under inert atmosphere using N₂ gas. Dry THF (AcrosOrganics), methanol (Acros Organics), pyridine (Acros Organics),ethylenediamine (Alfa Aesar), and all other reagents (Sigma-Aldrich)were used as supplied.

Example 1: Synthesis ofN-(2-aminoethyl)-2-(2-nitro-1H-imidazol-1-yl)acetamide

An oven-dried 50 mL round bottom flask was charged with a solution ofmethyl 2-(2-nitro-1H-imidazol-1-yl)acetate^([40]) (500 mg, 2.7 mmol) inmethanol (4.72 mL) and a magnetic stir bar. Ethylenediamine (0.722 mL,10.8 mmol) was added dropwise to this solution over 1 minute and themixture was allowed to stir at room temperature for 18 hours. Solventwas then removed via rotary evaporation and the resultant solid wasdried under vacuum for 2 days to afford 3 (575 mg, quantitative) as anamorphous pale yellow solid. ¹H NMR (500 MHz, MeOD): δ 7.45 (d, 1H,J=1.2 Hz, Ar), 7.17 (d, 1H, J=1.2 Hz, Ar), 5.17 (s, 2H, Ar—CH ₂—CO—),3.31 (t, 2H, J=6.2 Hz, —CH₂—CH ₂—NHCO—+residual MeOD overlap), 2.75 (t,2H, J=6.2 Hz, H₂N—CH ₂—CH₂—); ¹³C NMR (125 MHz, MeOD): δ 167.00, 128.00,126.97, 51.48, 41.74, 40.47. HRMS m/z calcd. for C₇H₁₂N₅O₃ (MH⁺)214.0940, found 214.0937.

Example 2: Synthesis of Compounds 1-5

3-methyltellanyl-1-ethanol (1)

Tellurium metal (granular, −5-+50 mesh, 500 mg, 3.9 mmol) was groundedto a fine powder using a mortar and pestle and suspended in THF (50 mL).Methyl lithium (2.5 mL, 4.0 mmol) was added drop-wise to the suspensionuntil the solution became a homogenous yellow solution at roomtemperature. The resulting mixture was cooled to −196° C. in a liquidnitrogen bath. Upon freezing, 2-chloro-ethanol (0.261 mL, 3.9 mmol) wasadded in one portion and the reaction was warmed to room temperature.The reaction mixture was stirred at room temperature for 2.5 hours. Oncethe reaction was complete by TLC, sat. NH₄Cl (100 mL) was added to themixture. The solution was extracted into diethyl ether (2×100 mL). Thecombined organic layer was washed with brine (1×100 mL), dried overMgSO₄, filtered and concentrated. The crude compound was purified bycolumn chromatography on silica gel (10% EtOAc in Pentane) and driedunder vacuum to give a viscous yellow oil. Yield: 66%, 488 mgs. ¹H NMR(500 MHz, CDCl₃, δ): 3.78 (s, —CH₂OH, 2H), 2.80 (t, J=6.8 Hz, —CH₂CH₂OH,2H), 1.88 (s, —TeCH₃, 3H); ¹³C NMR (125 MHz, CDCl₃, δ): 62.59 (—CH₂OH),8.40 (—CH₂—Te), −22.41 (—Te—CH₃). [M+NH₄]⁺=207.99829.

3-methyltellanyl-1-propanol (2)

Tellurium metal (granular, −5-+50 mesh, 500 mg, 3.9 mmol) was ground toa fine powder using a mortar and pestle and suspended in THF (50 mL).Methyl lithium (2.5 mL, 4.0 mmol) was added drop-wise to the suspensionuntil the solution turned yellow at room temperature. The resultingmixture was cooled to −196° C. in a liquid nitrogen bath. Upon freezing,1-chloro-3-propanol (0.326 mL, 3.9 mmol) was added in one portion andthe reaction was warmed to room temperature. The reaction mixture wasstirred at room temperature for 2.5 hours. Once the reaction wascomplete by TLC, sat. NH₄Cl (100 mL) was added to the mixture. Thesolution was extracted into diethyl ether (2×100 mL). The combinedorganic layer was washed with brine (1×100 mL), dried over MgSO₄,filtered, concentrated and dried under vacuum to give a viscous darkorange oil product. Yield: 74%, 581 mgs. Characterization equivalent tothat of literature. Angew. Chem. Int. Ed. Engl., 2014, 53, 11473-11477.

methyl 3-methyltellanyl-propionate (3)

Tellurium metal (granular, −5-+50 mesh, 500 mg, 3.9 mmol) was ground toa fine powder using a mortar and pestle and suspended in THF (50 mL).Methyl lithium (2.5 mL, 4.0 mmol) was added drop-wise to the suspensionuntil the solution turned yellow at room temperature. Water (0.18 mL)was added to the solution, inducing a color change to a dark brownmixture. The resulting mixture was cooled to −196° C. in a liquidnitrogen bath. Upon freezing, methyl acrylate (0.355 mL, 3.9 mmol) wasadded in one portion and the reaction was warmed to room temperature.The reaction mixture was stirred at room temperature for 0.5 hours. Oncethe reaction was complete by TLC, sat. NH₄Cl (100 mL) was added to themixture. The solution was extracted into diethyl ether (2×100 mL). Thecombined organic layer was washed with brine (1×100 mL), dried overMgSO₄, filtered, concentrated and dried under vacuum to give a viscousdark yellow oil. Yield: 85%, 775 mgs. ¹H NMR (500 MHz, CDCl₃, δ): 3.68(s, —COOCH₃, 3H), 2.86 (m, —CH₂COOCH₃, 2H), 2.76 (m, —CH₂CH₂Te—, 2H),1.92 (s, —Te—CH₃, 3H); ¹³C NMR (125 MHz, CDCl₃, δ): 173.87 (C═O), 52.11(—COOCH₃), 37.17 (—CH₂COOCH₃), −4.72 (—TeCH₂CH₂—), −21.35 (—Te—CH₃).[M+H]⁺=232.98149.

methyl 4-methyltellanyl-butanoate (4)

Tellurium metal (granular, −5-+50 mesh, 500 mg, 3.9 mmol) was ground toa fine powder using a mortar and pestle and suspended in THF (50 mL).Methyl lithium (2.5 mL, 4.0 mmol) was added drop-wise to the suspensionuntil the solution turned yellow at room temperature. The resultingmixture was cooled to −196° C. in a liquid nitrogen bath. Upon freezing,methyl-4-chlorobutyrate (0.478 mL, 3.9 mmol) was added in one portionand the reaction was warmed to room temperature. The reaction mixturewas stirred at room temperature for 2.5 hours. Once the reaction wascomplete by TLC, sat. NH₄Cl (100 mL) was added to the mixture. Thesolution was extracted into diethyl ether (2×100 mL). The combinedorganic layer was washed with brine (1×100 mL), dried over MgSO₄,filtered, concentrated and dried under vacuum to give a viscous darkyellow oil. Yield: 91%, 877 mgs. ¹H NMR (500 MHz, CDCl₃, δ): 3.64 (s,—COOCH₃, 3H), 2.60 (t, J=7.6 Hz, —TeCH₂-2H), 2.39 (t, J=7.4 Hz,—CH₂COOCH₃, 2H), 2.01 (m, —CH₂CH₂CH₂—, 2H), 1.86 (s, —TeCH₃, 3H); ¹³CNMR (125 MHz, CDCl₃, δ): 173.09 (C═O), 51.36 (—COOCH₃), 35.71(—CH₂C═O—), 26.76 (—CH₂CH₂C═O—), 1.92 (—TeCH₂CH₂—), −22.52 (—TeCH₃).[M+H]⁺=246.99690.

methyl 4-((trifluoromethyl)tellanyl)butanoate (5)

Tellurium metal (granular, −5-+50 mesh, 500 mg, 3.9 mmol) was ground toa fine powder using a mortar and pestle and suspended in 7 mL of DME.The solution was cooled to −60° C. using a 40% ethylene glycol 60%ethanol and dry ice cooling bath. Upon cooling,trimethyl(trifluoromethyl)silane (0.356 mL, 2.61 mmol) andtetramethylammonium fluoride (243 mg, 2.61 mmol) were added to thereaction mixture. The reaction was stirred vigorously for 1 hour at −60°C. and for 3 hours at room temperature. Once the reaction was complete,the yellow supernatant was decanted off and the solid residues remainingwere washed with DME. The supernatant and the washes were combined andconcentrated. To the concentrated crude mixture, 3 mL of DME and methyl4-bromobutyrate (0.230 mL, 1.82 mmol) were added. The reaction mixturewas stirred over-night at room temperature. Once the reaction wascomplete, the DME was removed by rotary-vaporization and the remainingcrude mixture was taken up in EtOAc. This organic layer was washed withwater (3×), brine (1×), dried over MgSO₄, filtered and concentrated. Thecrude mixture was purified by column chromatography (Toluene on silicagel). Yield: 50%, 270 mgs. ¹H NMR (500 MHz, CDCl₃, δ): 3.68 (s, —COOCH₃,3H), 3.13 (t, J=7.7 Hz, —TeCH₂—, 2H), 2.47 (t, J=7.7 Hz, —CH₂COOCH₃,−2H), 2.26 (p, J=7.1 Hz, —CH₂CH₂CH₂—, 2H); ¹³C NMR (125 MHz, CDCl₃, δ):172.82 (C═O), 103.79-95.40 (q, J=351.5 Hz, —Te—CF₃), 51.763 (—COOCH₃),35.41 (—CH₂CH₂CH₂Te—), 27.08 (—CH₂CH₂CH₂—), 8.20 (—TeCH₂—).[M+NH₄]⁺=317.99529.

Example 3: Synthesis of Compounds 6 and 7

1-(bromoethynyl)triisopropylsilane

N-bromosuccinimide (5.15 g, 29 mmol), silver nitrate (4.28 g, 25.2 mmol)and TIPS-acetylene (5.6 mL, 25.2 mmol) were added to 200 mL of acetone.The solution mixture was stirred vigorously for 3 hours at roomtemperature. Once the reaction was complete, 150 mL of water was addedto the mixture. The solution was extracted into hexane (3×125 mL). Thecombined organic layer was washed with brine (2×), dried over MgSO₄,filtered, concentrated and dried under vacuum to give a clear oilproduct. Yield: 6.51 g, 98%. Ref: Org. Lett. 2011, 13, 537-539.

hepta-4,6-diynoic acid intermediates

Cadiot-Chodkiewics coupling was completed according to literature. (J.P. Marino, H. N. Nguyen, J. Org. Chem., 2002, 67, 6841-6844.) CuCl (15mg, 0.15 mmol) was added to an aqueous solution of 30% BuNH₂ (25 mL) atroom temperature which generated a transparent blue solution. A fewhydroxylamine hydrochloride crystals were added to this solution mixtureto discharge the color. 4-pentynoic acid (901 mg, 9.2 mmol) was added tothe mixture at once, resulting in a yellow suspension. This solution wascooled using an ice-water bath. Upon cooling,2-bromo-1-triisopropylsilyl acetylene (2 g, 7.6 mmol) was addeddrop-wise. Additional crystals of hydroxylamine hydrochloride were addedto maintain the yellow solution when blue-green color changes occurred.The reaction was stirred vigorously for 0.5 hours. Once the reaction wascomplete by TLC, the solution was extracted with EtOAc (2×100 mL). Thecombined organic layer was washed with 1M HCl (1×100 mL), brine (1×100mL), dried with MgSO₄, filtered, concentrated and dried under vacuum togive a dark brown crude crystalline product (1.6 g, 76%). The crudeproduct of 6-(triisopropylsilyl)hepta-4,6-diynoic acid (388 mg, 1.39mmol) was dissolved in THF (15 mL). This solution mixture was cooledusing an ice-water bath. While cooling, tetrabutylammonium fluoride(1.39 mL, 1M in THF) was added dropwise until the solution reached roomtemperature. The reaction was stirred vigorously for 3 hours. Once thereaction was complete, the solution was extracted with EtOAc (3×100 mL).The combined organic layer was washed with 1M citric acid (3×100 mL),brine (3×100 mL), dried with MgSO₄, filtered, concentrated and driedunder vacuum to give a brown crude oil product. The compound was takendirectly to the next step of 2-(tellurophene-2-yl)propanoic acidsynthesis.

2-(tellurophen-2-yl)propanoic acid (6)

Tellurium metal (granular, −5-+50 mesh, 3.0 g, 12.68 mmol) was ground toa fine powder using a mortar and pestle. The tellurium powder was addedto an aqueous solution of 1M NaOH (30 mL). To the reaction mixture,sodium hydroxymethylsulfinate (6.0 g, 21.18 mmol) was added and stirredvigorously. The reaction solution was heated using an oil bath, to 95°C. for 0.5 hours and the solution turned a deep purple color. Thereaction solution was cooled to 60° C. and stirred for an additional 5mins. In 5 mL of ethanol, hepta-4,6-diynoic acid (388 mg, 3.17 mmol) wasadded to the reaction mixture. This solution mixture was stirred for 1.5hours at 60° C. The reaction was then exposed to oxygen by removing thesepta and allowing in atmosphere. The reaction was allowed to cool toroom temperature and stirred for 15 mins. Upon cooling, the reaction wasdiluted with a sat. NH₄Cl solution (100 mL). The solution was extractedwith EtOAc (2×100 mL). The combined organic layer was washed with 1M HCl(2×100 mL), brine (2×100 mL), dried over MgSO₄, filtered, concentrated,and dried under vacuum to give a dark yellow solid crude product. Thecrude product was purified by flash chromatography (5%-50% EtOAc/Hexaneson silica gel) to give a light yellow solid (563 mg, 70%). ¹H NMR (500MHz, CDCl₃, δ): 8.71 (dd, J=6.9, 1.2 Hz, —HCTe—, 1H), 7.59 (m, —HCHCTe—,1H), 7.38 (m, —TeCCH—, 1H), 3.22 (t, J=7.3, Tephene-CH₂—, 2H), 2.73 (t,J=7.3, —CH₂CH₂COOH, 2H); ¹³C NMR (125 MHz, CDCl₃, δ): 178.94 (C═O),148.69 (—TeCCH—), 137.42 (—HCHCTe—), 136.15 (—TeCCH—), 125.29 (—HCTe—),37.95 (Tephene-CH₂—), 32.01 (—CH₂CH₂COOH). [M+H]⁺=254.96665.

hexa-3,5-diyn-1-ol intermediate

Cadiot-Chodkiewics coupling. CuCl (7.5 mg, 0.08 mmol) was added to anaqueous solution of 30% BuNH₂ (25 mL) at room temperature that generateda transparent blue solution. A few hydroxylamine hydrochloride crystalswere added to this solution mixture to discharge the color. 3-Butyn-1-ol(1 g, 3.83 mmol) was added to the mixture resulting in a yellowsuspension. This solution was cooled using an ice-water bath. Uponcooling, 2-bromo-1-triisopropylsilyl acetylene (832 mg, 3.19 mmol) wasadded drop-wise. Additional crystals of hydroxylamine hydrochloride wereadded to prevent the solution from turning a blue-green color. Thereaction was stirred vigorously for 0.5 hours. Once the reaction wascomplete by TLC, the solution was extracted with EtOAc (2×100 mL). Thecombined organic layer was washed with 1M HCl (1×100 mL), brine (1×100mL), dried with MgSO4, filtered, concentrated and dried under vacuum togive a neat dark brown oil.

This product was directly deprotected to produce2-(tellurophen-2yl)ethan-ol. The product,6-(triisopropylsilyl)hexa-3,5-diyn-1-ol (873 mg, 1.44 mmol) wasdissolved in THF (15 mL). This solution mixture was cooled using anice-water (1:1) bath. Upon cooling, tetrabutylammonium fluoride (1.44mL, 1M in THF) was added dropwise and the solution was allowed to warmto room temperature. The reaction was stirred vigorously for 3 hours.Once the reaction was complete by TLC, the solution was extracted withEtOAc (3×100 mL). The combined organic layer was washed with 1 M citricacid (3×100 mL), brine (3×100 mL), dried with MgSO₄, filtered,concentrated and dried under vacuum to give a brown oil. This crudeproduct was immediately taken to the next step for the synthesis of2-(tellurophen-2yl)ethan-ol since the compound possess limited stabilityas a free diacetylene.

2-(tellurophen-2-yl)ethan-ol (7)

Tellurium metal (granular, −5-+50 mesh, 3.2 g, 41.96 mmol) was ground toa fine powder using a mortar and pestle. The tellurium powder was addedto an aqueous solution of 1M NaOH (30 mL). To the reaction mixture,sodium hydroxymethylsulfinate (6.4 g, 42.47 mmol) was added and stirredvigorously. The reaction solution was heated using an oil bath, to 95°C. for 0.5 hours and the solution turned a deep purple color. Thereaction solution was cooled to 60° C. and stirred for an additional 5mins. In ethanol (5 mL), hexa-3,5-diyn-1-ol (600 mg, 6.37 mmol) wasadded to the reaction mixture. This solution mixture was stirred for 1.5hours at 60° C. At this point, the reaction was exposed to oxygen byremoving the septa and exposing the reaction to atmosphere. The reactionwas cooled to room temperature and allowed to stir for 15 mins. Uponcooling, the reaction was diluted with a sat. NH₄Cl solution (100 mL).The solution was extracted with EtOAc (2×100 mL). The combined organiclayer was washed with 1M HCl (2×100 mL), brine (2×100 mL), dried withMgSO₄, filtered concentrated, and dried under vacuum to give a darkyellow oil crude product. The crude product was purified by flashchromatography (10%-30% EtOAc/Hexanes on silica gel stationary phase) togive a light yellow oil (949 mg, 66%). ¹H NMR (400 MHz, CDCl₃, δ): 8.71(dd, J=6.9, 1.2 Hz, —HCTe—, 1H), 7.65 (m, —HCHCTe—, 1H), 7.44 (m,—TeCCH—, 1H), 3.82 (t, J=6.0 Hz, Tephene-CH₂—, 2H), 3.13 (t, J=6.4 Hz,—CH₂CH₂OH, 2H), 2.68 (s, —OH, 1H). ¹³C NMR (100 MHz, CDCl₃): 145.37(—TeCCH—), 136.34 (—HCHCTe—), 135.48 (—TeCCH—), 124.89 (—HCTe—), 63.64(—CH₂CH₂OH), 38.85 (—Tephene-CH₂—). [M+H]⁺=254.96665

Example 4: Carbamylation of Compound 2 with Benzylamine

Example 5: Amidation of Compounds 4-6 with Benzylamine

N-benzyl-4-(methyltellanyl)butanamide (9)

Compound 4 (400 mg, 1.64 mmol) was dissolved in THF (25 mL) and stirredvigorously. To the mixture, 1 M NaOH (25 mL) was added and a biphasicmixture was generated. This solution was stirred for 1 hour. Thereaction was then diluted with H₂O (50 mL) and 1 M citric acid was addeduntil the reaction mixture was acidic by pH paper. The resulting mixturewas extracted into diethyl ether (3×100 mL) and washed with brine (2×100mL). The solvent was removed from the combined organic layers by rotaryevaporation. This compound was re-dissolved in DCM (5 mL) and added to anew round bottom flask where DCC (355 mg, 1.72 mmol) was added andstirred for 5 minutes. Once the mixture became a milky solution, NHS(198.2 mg, 1.72 mmol) was added to the mixture and stirred for anadditional 5 minutes. To this resulting solution, a mixture ofbenzylamine (215 μL, 1.97 mmol) and TEA (275 μL, 1.97 mmol) in DCM (5mL) were added at room temperature. The reaction was stirred overnight.Once the reaction was complete by TLC, the stir bar was removed and thesolvent was removed by rotary evaporation. The resulting product wasre-dissolved in cold EtOAc (100 mL) where white precipitate formed inthe solution. The precipitate, presumed to be DCU, was removed byfiltration. This process was repeated 3 times to remove the DCU. Thefiltrate was washed with 0.5 M citric acid (2×100 mL), NaHCO₃ (2×100 mL)and brine (1×100 mL). The organic layers were combined, dried withMgSO₄, filtered, concentrated and dried under vacuum to give a lightyellow solid product (440 mg, 81%). ¹H NMR (500 MHz, CDCl₃, δ): 7.33 (m,aryl-, 5H), 5.92 (s, —NH-1H), 4.40 (d, J=5.8 Hz, aryl-CH₂NH-2H), 2.62(t, J=7.4 Hz, —TeCH₂—, 2H), 2.29 (t, J=7.3 Hz, —COCH₂CH₂CH₂Te—, 2H),2.06 (m, —COCH₂CH₂CH₂Te—, 2H), 1.86 (s, —TeCH₃, 3H). ¹³C NMR (125 MHz,CDCl₃, δ): 172.25 (C═O), (138.63, 129.08, 128.17 & 127.89, aryl), 43.99(aryl-CH₂—NH—), 38.66 (—CH₂CH₂CH₂Te—), 27.78 (—CH₂CH₂CH₂Te—), 2.90(—CH₂TeCH₃), −21.95 (—TeCH₃). [M+H]⁺=322.04378.

N-benzyl-4-((trifluoromethyl)tellanyl)butamide (10)

Compound 5 (400 mg, 1.4 mmol) was dissolved in 25 mL of THF and stirredvigorously. To the mixture, 25 mL of 1 M NaOH was added and a biphasicmixture was generated. This solution was stirred for 1 hour. Uponcompletion, the reaction was diluted and 1 M citric acid was added untilthe reaction mixture was acidic. The resulting mixture was extractedinto ethyl acetate (3×100 mL) and washed with brine (2×100 mL). Thecombined organic layers were combined and the solvent was removed byrotary evaporation. This compound was re-dissolved in DCM (5 mL) andadded to a new round bottom flask where DCC (303 mg, 1.47 mmol) wasadded and stirred for 5 minutes. Once the mixture became a milkysolution, NHS (169 mg, 1.47 mmol) was added to the mixture and stirredfor an addition 5 minutes. To this resulting solution, a mixture ofbenzylamine (180 μL, 1.68 mmol) and TEA (235 μL, 1.68 mmol) in 5 mL ofDCM was added at room temperature. The reaction was stirred overnight.Upon completion, the stir bar was removed and the solvent was removed byrotary evaporation. The resulting product was redissolved in cold EtOAc(100 mL) where white precipitate formed. The precipitate, presumed to beDCU, was removed by filtration. The filtrate was washed with 0.5 Mcitric acid (2×100 mL), NaHCO₃ (2×100 mL) and brine (1×100 mL). Theorganic layers were combined, dried with MgSO₄, filtered, concentratedand dried under vacuum to give a yellow solid product (157 mg, 30%). ¹HNMR (500 MHz, CDCl₃, δ): 7.33 (m, aryl-, 5H), 5.79 (s, —NH-1H), 4.41 (d,J=5.7 Hz, aryl-CH₂NH—, 2H), 3.14 (t, J=6.9 Hz, —CH₂Te—, 2H), 2.34 (m,—CH₂CH₂CH₂Te—, 2H), 2.28 (p, J=6.8, —CH₂CH₂CH₂Te—, 2H). ¹³C NMR (125MHz, CDCl₃, δ): 171.87 (C═O), (138.40, 129.19, 128.26 & 128.07, aryl),(104.84-96.44, —CF₃), 44.15 (aryl-CH₂—NH—), 37.94 (—CH₂CH₂CH₂Te—), 27.75(—CH₂CH₂CH₂Te—), 9.05 (—CH₂CH₂CH₂Te—). [M+H]⁺=376.0175.

N-benzyl-3-(tellurophen-2-yl)propanamide (11)

Compound 6 (100 mg, 0.4 mmol) was dissolved in dissolved in DCM (5 mL)and DCC (86 mg, 0.42 mmol) was added and stirred for 5 minutes. Once themixture became a milky solution, NHS (48 mg, 0.42 mmol) was added andstirred for an addition 5 minutes. To this resulting solution, a mixtureof benzylamine (52 μL, 0.48 mmol) and TEA (67 μL, 0.48 mmol) in 5 mL ofDCM was added at room temperature. The reaction was stirred overnight.Upon completion, the stir bar was removed and the solvent was removed byrotary evaporation. The resulting product was redissolved in cold EtOAc(150 mL) where white precipitate crashed out of solution. Theprecipitate, presumed to be DCU, was removed by filtration and thefiltrate was washed with 0.5 M citric acid (2×150 mL), NaHCO₃ (2×150 mL)and brine (1×150 mL). The organic layers were combined, dried withMgSO₄, filtered, concentrated and dried under vacuum to give a yellowsolid. The product was purified by flash chromatography (5%-25%EtOAc/Hexanes) to give a yellow solid (440 mg, 81%). ¹H NMR (500 MHz,CDCl₃, δ): 8.71 (dd, J=6.9, 1.3 Hz, —HCTe—, 1H), 7.57 (m, —HCHCTe—, 1H),7.31 (m, —TeCCH— & aryl, 7H), 5.76 (s, —NH—, 1H), 4.41 (d, J=5.7 Hz,aryl-CH₂NH—, 2H), 3.25 (t, J=7.6 Hz, Tephene-CH₂CH₂—, 2H), 2.53 (t,J=7.6 Hz, Tephene-CH₂CH₂—, 2H). ¹³C NMR (125 MHz, CDCl₃, δ): 171.31(C═O), 148.96 (—TeCCH—), 137.85 (—HCHCTe—), 136.73 (—TeCCH—), (135.54,128.84, 128.56, 127.7, 127.39 & 124.87, aryl), 43.58 (aryl-CH₂NH—),39.88 (Tephene-CH₂CH₂—), 32.33 (Tephene-CH₂CH₂—). [M+H]⁺=344.02941

Example 6: 2-(tellurophen-2yl)methanol (12)

2-(tellurophen-2yl)methanol (12)

Tellurium metal (granular, −5-+50 mesh, 640 mg, 5 mmol) was ground to afine powder using a mortar and pestle. The tellurium powder was added toan aqueous solution of 1M NaOH (30 mL). To the reaction mixture, sodiumhydroxymethylsulfinate (1.18 g, 10 mmol) was added and stirredvigorously. The reaction solution was heated using an oil bath, to 95°C. for 0.5 hours and the solution turned a deep purple color. Thereaction solution was cooled to 60° C. and stirred for an additional 5mins. In 5 mL of ethanol, penta-2,4-diyn-1-ol (c) (100 mg, 1.25 mmol)was added to the reaction mixture. Compound c was prepared analogouslyto compound 6. This solution mixture was stirred for 1.5 hours at 60° C.The reaction was then exposed to oxygen by removing the septa andallowing in atmosphere. The reaction was allowed to cool to roomtemperature and stirred for 15 mins. Upon cooling, the reaction wasdiluted with a sat. NH₄Cl solution (100 mL). The solution was extractedwith EtOAc (2×100 mL). The combined organic layer was washed with 1M HCl(2×100 mL), brine (2×100 mL), dried over MgSO₄, filtered, concentrated,and dried under vacuum to give a dark orange-brown oil crude product.The crude product was purified by flash chromatography (5%-50%EtOAc/Hexanes on silica gel) to give a light yellow solid (200 mg, 76%).¹H NMR (500 MHz, CDCl₃, 5): 8.83 (dd, J=6.9, 1.2 Hz, —HCTe—, 1H), 7.68(m, —HCHCTe—, 1H), 7.49 (m, —TeCCH—, 1H), 4.83 (d, J=1.2 Hz,Tephene-CH₂—, 2H).

Example 7: 2-(chloromethyl)tellurophene (13)

2-(chloromethyl)tellurophene (13)

2-(tellurophen-2yl)methanol (100 mg, 0.47 mmol) and triphenylphosphine(156 mg, 0.59 mmol) was added to solution of acetonitrile (15 mL).Carbon tetrachloride (300 uL, 0.47 mmol) was added drop-wise to thesolution. The reaction was refluxed at 80° C. for 30 mins. The reactionwas allowed to cool to room temperature and stirred for 5 mins. Uponcooling, the reaction was concentrated and dried under vacuum to give anorange oil crude product (55 mg, 41%). ¹H NMR (400 MHz, CDCl₃, δ): 7.61(m, —HCTe—, 1H), 7.48 (m, —HCHCTe—, 1H), 7.41 (m, —TeCCH—, 1H), 4.74 (d,J=1.1 Hz, Tephene-CH₂—, 2H).

Example 8: 2,5-dioxopyrrolidin-1-yl 3-(tellurophen-2-yl)propanoate (14)

2,5-dioxopyrrolidin-1-yl 3-(tellurophen-2-yl)propanoate (14)

3-(tellurophen-2-yl)propanoic acid (150 mg, 0.59 mmol) was dissolved ina solution mixture of EtOAc (5 mL) and pyridine (2.5 mL). T3P (379 mg,1.18 mmol) was added to the mixture and the reaction was cooled to 0° C.using an ice bath and stirred for 5 mins. Upon cooling, NHS (75 mg, 0.65mmol) was added to the reaction and the reaction was allowed to reachroom temperature. The reaction was stirred overnight at roomtemperature. Upon completion by TLC, the reaction was diluted with water(50 mL). The resulting reaction mixture was extracted into EtOAc (50mL×3). The combined organic layers were concentrated and dried undervacuum to give the product (155 mg, 76%). %). ¹H NMR (500 MHz, CDCl₃,δ): 8.73 (dd, J=6.9, 1.2 Hz, —HCTe—, 1H), 7.59 (m, —HCHCTe—, 1H), 7.41(m, —TeCCH—, 1H), 3.33 (t, J=7.3, Tephene-CH₂—, 2H), 2.97 (t, J=7.3,—CH₂CH₂COOH, 2H), 2.84 (s, succinimide, 4H).

Example 9:(S)-2-((tert-butoxycarbonyl)amino)-7-(triisopropylsilyl)hepta-4,6-diynoicacid (15)

(S)-2-((tert-butoxycarbonyl)amino)-7-(triisopropylsilyl)hepta-4,6-diynoicacid (15)

A scintillation vial was charged with CuCl (19 mg, 0.1914 mmol), aqueousn-butylamine (5.5 mL, 30% n-butylamine: H₂O (v/v)), and a magnetic stirbar. Several grains of hydroxylamine hydrochloride were added to thevigorously-stirring blue solution until the solution turned clear. Next,boc-L-propargylglycine (free acid, 428.5 mg, 2.01 mmol) was addedquickly, the atmosphere of the vial was exchanged with argon, and thevial cooled in an ice bath. To the resultant yellow solution was added(bromoethynyl)triisopropylsilane 1, (500 mg, 1.914 mmol), dropwise, over5 minutes. After this addition was complete the ice bath was removed andthe reaction was allowed to stir for at least 4 hours at roomtemperature. If the reaction turned blue, additional grains ofhydroxylamine hydrochloride were added; this reverted the solution backto a yellow/reddish-brown. Once the reaction was complete, the productwas extracted into diethylether (3× wash with 1.0 M HCl), dried overanhydrous MgSO₄, and concentrated to afford the title compound as aviscous clear oil (750 mg, quantitative).

Example 10:(S)-2-((tert-butoxycarbonyl)amino)-3-(tellurophen-2-yl)propanoic acid(16)

(S)-2-((tert-butoxycarbonyl)amino)-3-(tellurophen-2-yl)propanoic acid(16)

Part A:

An oven-dried 25 mL round bottom flask was charged with(S)-2-((tert-butoxycarbonyl)amino)-7-(triisopropylsilyl)hepta-4,6-diynoicacid (750 mg, 1.9 mmol), dry tetrahydrofuran 15 (7.7 mL), and a drymagnetic stir bar. The flask was then cooled on an ice bath and theatmosphere exchanged with argon. Tetrabutylammonium fluoride (7.7 mL ofa 1.0 M solution in anhydrous tetrahydrofuran, 7.6 mmol) was then addedall at once. The reaction was allowed to stir on ice for 15 minutes,after which the entire mixture was injected all at once into thesolution prepared in part B.

Part B:

A 2-neck 250 mL round bottom flask was charged with monosodiumhydroxymethanesulfinate dihydrate (3.26 g, 21.12 mmol),freshly-pulverized tellurium metal (from −5-+50 mesh pellets, 270 mg,2.11 mmol), degassed aqueous sodium hydroxide (2.0 M, 20 mL), absoluteethanol (20 mL), and a magnetic stir bar. Argon gas was bubbled throughthe solution for 20 minutes with stirring. One neck of the reactionvessel was then fitted with a reflux condenser, and the other with arubber septum. The vessel was then placed in a silicon oil bath at 75°C. and a constant stream of argon gas was maintained flowing through theflask. Once a deep purple solution formed, the temperature of the oilbath was lowered to 40° C. Once the contents of the flask equilibratedwith the new temperature of the oil bath, the solution prepared in PartA was injected all at once through the rubber septum. The reaction wasallowed to stir for at least 4 hours, after which the flask was removedfrom the oil bath, the contents exposed to air until unreacted telluriummetal precipitated, and the organic components extracted into ethylacetate (5-10× wash with 1.0 M HCl), dried over anhydrous MgSO₄, andconcentrated to afford the title compound as an impure yellow oil. Theproduct was further purified via flash chromatography (silica gelstationary phase, 1% triethylamine, 3% methanol, 96% chloroform mobilephase, product R_(f)˜0.55-0.6 on silica-coated thin layer chromatographyplate with 2% triethylamine, 8% methanol, 90% chloroform mobile phase,staining with KMnO₄) to afford the triethylammonium salt of the titlecompound as a viscous clear oil (474 mg, 53% as calculated from the(bromoethynyl)triisopropylsilane starting material). ¹H NMR (400 MHz,CDCl₃): δ 8.60 (dd, 1H, J=7.0, 1.2 Hz), 7.54 (dd, 1H, J=7.0, 3.9 Hz),7.35 (m, 1H), 5.65 (br s, 1H), 4.26 (br s, 1H), 3.48 (br s, 2H), 1.41(s, 9H) ppm.

Example 11: (S)-2-amino-3-(tellurophen-2-yl)propanoic acidtriethylammonium salt (17)

(S)-2-amino-3-(tellurophen-2-yl)propanoic acid triethylammoinum salt(17)

An oven-dried scintillation vial in an ice bath was charged with thetriethylammonium salt of(S)-2-((tert-butoxycarbonyl)amino)-3-(tellurophen-2-yl)propanoic acid(16) (234 mg, 0.5 mmol), dichloromethane (2.5 mL), trifluoroacetic acid(2.5 mL), and a dry magnetic stir bar. The reaction was allowed to stirin the ice bath for 40 minutes, after which the reaction was neutralizedwith triethylamine (monitored using litmus paper). The reaction was thenconcentrated, and reconstituted in a small volume of 2% methanol/H₂O.The product was purified on a C18 reverse-phase plug (2% methanol/H₂O to1:1 methanol/H₂O mobile phase) to afford the title compound (100 mg,54%) as a clear oil which solidified (white solid) upon freeze-drying.¹H NMR (400 MHz, MeOD-d₄): δ 8.92 (dd, 1H, J=7.0, 1.3 Hz), 7.63 (dd, 1H,J=7.0, 3.9 Hz), 7.52 (dd, 1H, J=3.9, 1.3 Hz), 4.21 (app t, 1H, J=5.0Hz), 3.49 (m, 2H) ppm.

Example 12:(3S,4R,5S,6R)-6-(acetoxymethyl)-3-(3-(tellurophen-2-yl)propanamido)tetrahydro-2H-pyran-2,4,5-triyltriacetate (18)

(3S,4R,5S,6R)-6-(acetoxymethyl)-3-(3-(tellurophen-2-yl)propanamido)tetrahydro-2H-pyran-2,4,5-triyltriacetate (18)

A scintillation vial was charged with mannosamine hydrochloride (42.5mg, 0.197 mmol), aqueous sodium bicarbonate (3 mL, 100 mM),tetrahydrofuran (2 mL), 2,5-dioxopyrrolidin-1-yl3-(tellurophen-2-yl)propanoate (12) (86 mg, 0.247 mmol), and a magneticstir bar. The mixture was allowed to stir at room temperature for 18hours, after which the reaction was concentrated via rotary evaporationand further dried under high vacuum. Next, the dry reaction crude wasreconstituted in anhydrous pyridine (4 mL) and cooled in an ice bath.Acetic anhydride (3 mL) was then added and the mixture was allowed towarm to room temperature over 18 hours (with stirring). Volatilecompounds were removed via rotary evaporation (toluene was added to aidin evaporation of the pyridine). The product was purified via flashchromatography (silica gel stationary phase, 3:1 to 1:1 pentanes:ethylacetate, product Rf˜0.55 on silica-coated thin layer chromatographyplate with 1:1 pentanes:ethyl acetate mobile phase, staining withninhydrin) to afford the product as a clear oil (61 mg, 53%). ¹H NMR(500 MHz, CDCl₃): δ 8.71 (dd, 1H, J=6.9, 1.3 Hz), 7.58 (dd, 1H, J=6.9,3.8 Hz), 7.39 (m, 1H), 5.96 (d, 1H, J=1.9 Hz), 5.84 (br m, 1H), 5.31(dd, 1H, J=10.2, 4.6 Hz), 5.10 (t, 1H, J=10.2 Hz), 4.64 (ddd, 1H, J=9.1,4.6, 1.9 Hz), 4.24 (m, 1H), 4.03 (m, 2H), 3.24 (dt, 2H, J=6.9, 1.2 Hz),2.60 (m, 2H), 2.17 (s, 3H), 2.08 (s, 3H), 2.05 (s, 3H), 1.96 (s, 3H)ppm.

Example 13:(4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-(tellurophen-2-yl)propanamido)propyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylicacid sodium salt (19)

(4S,5R,6R)-5-acetamido-6-((1R,2R)-1,2-dihydroxy-3-(3-(tellurophen-2-yl)propanamido)propyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylicacid sodium salt (19)

A 25 mL round bottom flask was charged with(4S,5R,6R)-5-acetamido-6-((1R,2R)-3-amino-1,2-dihydroxypropyl)-2,4-dihydroxytetrahydro-2H-pyran-2-carboxylicacid (108 mg, 0.35 mmol), saturated aqueous sodium bicarbonate (5.9 mL),1,4-dioxane (5.8 mL), 2,5-dioxopyrrolidin-1-yl3-(tellurophen-2-yl)propanoate (12) (162.37 mg, 0.465 mmol), and amagnetic stir bar. The mixture was allowed to stir at room temperaturefor 18 hours, after which the reaction was concentrated via rotaryevaporation. The crude mixture was then reconstituted in 2% methanol/H₂Oand the product purified on a C18 reverse-phase plug (2% methanol/H₂O to1:1 methanol/H₂O mobile phase) to afford the title compound as a clearoil. ¹H NMR (500 MHz, MeOD-d₄): δ 8.70 (dd, 1H, J=6.9, 1.3 Hz), 7.52(dd, 1H, J=6.9, 3.9 Hz), 7.34 (m, 1H), 3.97 (m, 3H), 3.68 (m, 1H), 3.63(dd, 1H, J=13.8, 3.2 Hz), 3.17 (m, 3H), 2.53 (t, 2H, J=7.3 Hz), 2.08(dd, 1H, J=12.6, 4.4 Hz), 1.98 (s, 3H), 1.88 (t, 1H, J=11.8 Hz) ppm.NOTE: the missing resonance corresponding to a single proton may lieunder the residual solvent peak at 3.31 ppm.

Example 14:1-(2-difluoromethyl-4-(3-(tellurophen-2-yl)propanamido)phenyl)-β-D-galactopyranose(24)

Scheme 13.

Synthesis of B-galactosidase tellurophene probe (24). Reactions andconditions: a) Bu₄NBr, 1 M NaOH:DCM (52%); b) dimethylaminosulfurtrifluoride, DCM (89%); c) H₂, Pd/C, EtOAc (97%); d)3-(tellurophen-2-yl)propanoic acid, T3P,

pyridine:EtOAc (59%); e) NaOMe, MeOH (75%).

1-(2-formyl-4-nitrophenyl)-2-3,4,6-tetraacetyl-β-D-galactopyranose (20)

A solution of tetrabutylammonium bromide (0.783 g, 2.43 mmol) in 1 MNaOH (3.7 mL) was added to 2-hydroxy-5-nitrobenzaldehyde (0.609 g, 3.65mmol) in DCM (7.4 mL) with stirring at room temperature. A solution of2,3,4,6-tetraacetyl-α-D-galactopyranosyl bromide (1.00 g, 2.43 mmol) inminimal DCM was added, and the mixture was stirred for three days atroom temperature. It was subsequently diluted with DCM (200 mL) andwashed with 2 M NaOH (4×200 mL) and brine (200 mL). The organic extractwas dried over MgSO₄, filtered and concentrated to yield an orangesolid. The crude product was purified via flash chromatography(stationary phase, silica gel; mobile phase, DCM, 0%-5% MeOH, 0.1%triethylamine) to afford compound 20 (1.23 g, 51%) as a viscous orangeliquid. ¹H NMR (500 MHz, CDCl₃) δ 10.33 (s, 1H, CHO), 8.71 (d, J=3.0 Hz,1H, Ar—H), 8.42 (dd, J=9.0 Hz, 3.0 Hz, 1H, Ar—H), 7.25 (d, J=9.0 Hz, 1H,Ar—H), 5.61 (dd, J=10.5 Hz, 8.0 Hz, 1H, H-2), 5.51 (dd, J=3.5 Hz, 1.0Hz, 1H, H-4), 5.28 (d, J=7.5 Hz, H-1), 5.18 (dd, J=10.5 Hz, 3.5 Hz, 1H,H-3), 4.15-4.25 (m, 3H, H-5H-6a, H-6b), 2.20, 2.08, 2.07, 2.03 (s, 3H,4×COCH ₃).

1-(2-difluoromethyl-4-nitrophenyl)-2-3,4,6-tetraacetyl-β-D-galactopyranose(21)

Dimethylaminosulfur trifluoride (0.146 mL, 1.50 mmol) was added to asolution of 20 (0.622 g, 1.25 mmol) in dry DCM (16 mL). The reaction wasstirred at room temperature under N₂ for 6.5 h, then quenched by theaddition of ice (100 mL) and extracted into DCM (2×100 mL). The combinedorganic extracts were washed with water (100 mL) and brine (100 mL),dried over MgSO₄, filtered and concentrated to yield a yellow oil. Thecrude product was purified via flash chromatography (stationary phase,silica gel; mobile phase, DCM, 5% MeOH, 0.1% triethylamine) to affordcompound 21 (0.577 g, 89%) as a viscous yellow liquid. ¹H NMR (500 MHz,CDCl₃) δ 8.49 (dd, J=3.0 Hz, 1.5 Hz, 1H, Ar—H), 8.33 (dd, J=9.0 Hz, 2.0Hz, 1H, Ar—H), 7.22 (d, J=9.5 Hz, 1H, Ar—H), 6.85 (t, J=54.5 Hz, 1H,CHF₂), 5.57 (dd, J=10.5 Hz, 8.0 Hz, 1H, H-2), 5.50 (app d, J=3.5 Hz, 1H,H-4), 5.16 (d, J=8.0 Hz, H-1), 5.15 (dd, J=11.0 Hz, 3.5 Hz, 1H, H-3),4.15-4.25 (m, 3H, H-5H-6a, H-6b), 2.20, 2.09, 2.06, 2.03 (s, 3H,4×COCH₃). DART-MS m/z calcd. for C₂₁H₂₃F₂NO₁₂ 519.40, found 537.15170[M+NH₄]⁺.

1-(2-difluoromethyl-4-aminophenyl)-2-3,4,6-tetraacetyl-β-D-galactopyranose(22)

Pd/C (5% Pd, 0.220 g) was added to a stirring solution of 21 (1.08 g,2.08 mmol) in ethyl acetate (5 mL) in a 25 mL three-necked round-bottomflask. The flask was purged with N₂, then H₂, then placed under 2.04 atmof fresh H₂ overnight at room temperature with stirring. Pd/C wasfiltered through celite, and the filtrate was concentrated to affordpure 22 (0.990 g, 97%) as an orange solid. ¹H NMR (400 MHz, CDCl₃) δ6.94 (s, 1H, Ar—H), 6.87 (dd, J=2.8 Hz, 1.6 Hz, 1H, Ar—H), 6.80 (t,J=55.6 Hz, 1H, CHF₂), 5.47 (dd, J=10.8 Hz, 8.0 Hz, 1H, H-2), 5.44 (dd,J=3.2 Hz, 0.8 Hz, 1H, H-4), 5.08 (dd, J=10.8 Hz, 3.6 Hz, 1H, H-3), 4.86(d, J=8.0 Hz, 1H, H-1), 4.00-4.26 (m, 3H, H-5, H-6a, H-6b), 2.19, 2.08,2.06, 2.01 (s, 3H, 4×COCH₃ ). ¹⁹F NMR (376 MHz, CDCl₃) 5-108.43 (dd,J=300.8 Hz, 56.4 Hz, 1F), −122.67 (dd, J=300.8 Hz, 56.4 Hz, 1F). DART-MSm/z calcd. for C₂₁H₂₅F₂NO₁₀ 489.14, found 490.2 [M+H]⁺.

1-(2-difluoromethyl-4-(3-(tellurophen-2-yl)propanamido)phenyl)-2-3,4,6-tetraacetyl-β-D-galactopyranose(23)

22 (0.176 g, 0.360 mmol), 3-(tellurophen-2-yl)propanoic acid (6) (0.082g, 0.327 mmol), pyridine (0.10 mL) and ethyl acetate (0.20 mL) wereadded to a 25 mL round-bottom flask at −20° C. under N₂.Propylphosphonic acid (T3P, 50 wt. % in ethyl acetate, 0.43 mL) wasadded dropwise and the solution was stirred at 0° C. for 20 h. Thesolution was diluted with DCM (40 mL) and washed with saturated sodiumbicarbonate (3×40 mL), water (40 mL) and brine (40 mL). The organicextract was dried over MgSO₄, filtered and concentrated to yield ayellow solid. The crude product was purified via column chromatography(stationary phase, silica gel; mobile phase, DCM, 5% MeOH, 0.1%triethylamine) to afford 23 (0.153 g, 59%) as a yellow solid. ¹H NMR(400 MHz, CDCl₃) δ 8.64 (dd, J=6.8 Hz, 1.2 Hz, 1H, TeAr—H), 7.96 (app.s, 1H, TeAr—H), 7.76 (dd, J=9.2 Hz, 0.4 Hz, 1H, Ar—H), 7.53 (dd, J=10.8Hz, 4.0 Hz, 1H, TeAr—H), 7.45 (br s, 1H, NH), 7.33 (d, J=2.8 Hz, 1H,Ar—H), 7.04 (d, J=9.2 Hz, 1H, Ar—H), 6.77 (t, J=55.2 Hz, 1H, —CHF₂),5.47 (dd, J=10.8 Hz, 8.0 Hz, 1H, H-2), 5.44 (app. d, J=2.8 Hz, 1H, H-4),5.09 (dd, J=10.4 Hz, 3.2 Hz, 1H, H-3), 4.95 (d, J=8.0 Hz, 1H, H-1),4.04-4.22 (m, 3H, H-5, H-6a, H-6b), 3.25 (t, J=6.8 Hz, 2H, —OC—CH₂—CH₂), 2.64 (t, J=6.8 Hz, 2H, —CH₂—CH₂ —CTe), 2.15, 2.03, 2.02, 1.98 (s,3H, 3×-COCH₃ ). DART-MS m/z calcd. for C₂₈H₃₁F₂NO₁₁ ¹³⁰Te 725.09, found726.1 [M+H]⁺.

1-(2-difluoromethyl-4-(3-(tellurophen-2-yl)propanamido)phenyl)-β-D-galactopyranose(24)

23 (0.076 g, 0.106 mmol) was dissolved in dry methanol (2 mL) and a 0.5M solution of NaOMe in methanol (0.20 mL) was added dropwise to thestirring solution. After 3 h, the reaction was quenched by the additionof Dowex® 50WX2 hydrogen form resin (50-100 mesh) until neutral pH, andthe solution was concentrated to yield a pale yellow solid. The crudeproduct was desalted using a reverse-phase cartridge (stationary phase,C18; mobile phase, H₂O, 50%-100% MeOH) to yield 24 (0.044 g, 75%) as apale yellow solid. ¹H NMR (500 MHz, CD₃OD) δ 8.71 (dd, J=6.8 Hz, 1.2 Hz,1H, TeAr—H), 7.77 (d, J=2.5 Hz, 1H, TeAr—H), 7.63 (dd, J=9.0 Hz, 2.0 Hz,1H, Ar—H), 7.54 (dd, J=7.0 Hz, 4.0 Hz, 1H, TeAr—H), 7.39 (dd, J=4.0 Hz,1.5 Hz, 1H, Ar—H), 7.27 (s, 1H, Ar—H), 7.16 (t, J=55.5 Hz, 1H, —CHF₂)4.83 (d, J=8.0 Hz, 1H, H-1), 3.56-3.90 (m, 6H, H-2, H-3, H-4, H-5, H-6a,H-6b), 3.27 (t, J=8.0 Hz, 2H, —OC—CH₂ —CH₂), 2.69 (t, J=7.0 Hz, 2H,—CH₂—CH₂ —CTe). ¹³C NMR (126 MHz, CD₃OD) δ 171.58, 148.43, 136.07,135.13, 133.49, 124.14, 123.54, 117.29, 116.75, 112.97, 111.11, 109.24,102.64, 75.70, 73.43, 70.77, 68.76, 60.94, 39.81, 31.91. DART-MS m/zcalcd. for C₂₀H₂₃F₂NO₇ ¹³⁰Te 557.05, found 575.08786 [M+NH₄]⁺.

Example 15: Synthesis of Telox and Telox-2

Telox was made as described in Scheme 1 of U.S. Application Ser. No.62/039,762. The structure of Telox is as follows:

A tellurophene-containing hypoxia probe was accessed through thesynthetic pathway described in Scheme 14. This molecule is referred toherein as Telox-2.

Telox2 synthesized using 2-(tellurophen-2-yl)propanoic acid as astarting material is described below.

2-nitro-1-(2-(tellurophen-2-yl)ethyl)-1H-imidazole (Telox 2) (25)

An oven-dried 50 mL round bottom flask was charged with2-(tellurophen-2-yl)ethan-1-ol (7) (500 mg, 2.23 mmol), drytetrahydrofuran (15 mL), triphenylphosphine (1.17 g, 4.47 mmol),azomycin (505.4 mg, 4.47 mmol), and a dry magnetic stir bar. Thereaction vessel was flushed with nitrogen and cooled on an ice bath,Diisopropyl azodicarboxylate (0.822 mL, 4.47 mmol) was then addeddropwise over 5 minutes and the reaction was allowed to stir, warminggradually to room temperature, for 18 hours. The reaction was thenconcentrated via rotary evaporation and the product directly purifiedvia flash chromatography (silica gel stationary phase, 10-50%EtOAc/Pentanes, product R_(f)˜0.5 on silica-coated thin layerchromatography plate with 1:1 Pentanes/EtOAc mobile phase, staining withKMnO₄) to afford the title compound (674.2 mg, 95%) as a yellow solid.¹H NMR (600 MHz, CDCl₃): δ 8.79 (dd, 1H, J=6.6, 1.2 Hz), 7.55 (dd, 1H,J=7.2, 4.2 Hz), 7.24 (m, 1H), 7.07 (d, 1H, J=1.0 Hz), 6.93 (d, 1H, J=1.0Hz), 4.65 (t, 2H, J=7.0 Hz), 3.41 (dt, 2H, J=7.0, 1.1 Hz) ppm.

2-nitro-1-(2-(tellurophen-2-yl-5-d)ethyl)-1H-imidazole (Telox 2-d, 26)

An oven-dried 20 mL scintillation vial was charged with2-nitro-1-(2-(tellurophen-2-yl)ethyl)-1H-imidazole (Telox 2, 25, 25 mg,0.0784 mmol), methanol-d₄ (1 mL), deuterium oxide (1 mL),trifluoroacetic acid-d (0.308 mL, 4 mmol), and a magnetic stir bar. Thereaction was stirred under argon atmosphere for 18 hours at roomtemperature. The product was extracted out of solution into chloroform(3× wash with neutral deionized water), dried over anhydrous MgSO₄, andconcentrated, and obtained as a yellow solid (25 mg, quantitative). ¹HNMR (400 MHz, CDCl₃): δ 7.54 (d, 1H, J=3.84 Hz), 7.24 (dt, 1H, J=3.84,1.1 Hz), 7.06 (d, 1H, J=1.1 Hz), 6.92 (d, 1H, J=1.1 Hz), 4.63 (t, 2H,J=6.9 Hz), 3.40 (dt, 2H, J=6.9, 1.1 Hz) ppm.

Example 16

i. Cell Culture and Maintenance

The HCT116, colorectal carcinoma cell line (CCL-247™) was obtained fromAmerican Type Culture Collection and cultured/maintained in RPMI 1640medium supplemented with 10% fetal bovine serum.

ii. Hypoxia Exposure

HCT116 (500,000) cells were seeded in 60 mm plastic petri dishes(Corning Inc., NY) and incubated for 24 h at 37° C., 21% O₂/5% CO₂. Thecells were transferred to hypoxia chambers (H35/H85 hypoxia workstation,Don Whitley Scientific) maintained at 1%/0.2% or <0.02% O₂ for 3 h.Hypoxia experiments (<0.02% O₂) were performed by seeding the cells in60 mm glass plates (Corning Inc., NY).

iii. Confluency (Proliferative Toxicity) Assay

HCT116 cells (25,000) were seeded in a 24 well plate and incubatedovernight to allow the cells to adhere. The medium was removed and freshmedium with 50-400 μM TELOX or Telox2 was added and incubated for 1 h.The cells were then transported to a INCUCYTE™ Kinetic Imaging Systemthat was maintained either at 21% or 0.2% 02 at 37° C. Growth profileswere monitored by 10× objective every 4 h by IncuCyte™ ZOOM controlsoftware, using integrated confluence algorithm, until thecontrol-untreated cells reached stationary phase. Sixteen highdefinition-quality images per well were collected in phase-contrast modeand averaged to provide a representative statistical measure of the wellconfluence.

iv. Metabolic Toxicity Assay

A 96 well clear fluorometer plate was loaded with 200 μL of Jurkat cellsat a culture density of 1×10⁶ cells per mL. To each well was added anappropriate amount of a stock solution of Telox/Telox2 in sterile DMSOto reach a desired concentration of Telox/Telox2 (1-1000 micromole). Theconcentration of DMSO was 1% in all wells. Cells were allowed toincubate for 24 h at 37° C. under normal atmosphere, after which 20 μLof a commercially available solution of WST-1 in PBS (Roche Diagnostics,product #05015944001) was added to each well (gentle pipetting evenlydistributed the reagent throughout the well). Cells were allowed toincubate for a further 0.5 h at 37° C. under normal atmosphere, followedby subsequent measurement of the absorbance of each well at 450 nm usinga TECAN Safire 2 plate reader. Data was background corrected vs. wellsthat contained cell growth media (without cells), an appropriateconcentration of Telox2, a final concentration of DMSO=1%, and WST-1.Background correction wells were incubated in the same manner asdescribed for cell-positive wells.

v. Xanthine Oxidase Assay

A septa-sealable quartz cuvette was charged with K₂PO₄ buffer (800 μL,100 mM, pH 7.4), xanthine in K₂PO₄ buffer (100 μL, 5.0 mM xanthine(saturated solution), 100 mM buffer, pH 7.4), and either pimonidazole,Telox or Telox2 in K₂PO₄ buffer (100 μL, 1.0 mM of the 2-NI, 100 mMbuffer, pH 7.4). The cuvette was then sealed with a rubber septum andthe entire solution was degassed with high purity helium gas. Xanthineoxidase (0.2 units of grade III enzyme in a (NH₄)₂SO₄ suspension,Sigma-Aldrich, lot # SLBB1572V) was then added via Hamilton syringe andthe change in absorbance over time at 325 nm was recorded using anAgilent ultravioletvisible photospectrometer (model #8453).

vi. Traditional ICP-MS Experiments

HCT 116 cells (see sections i and ii) were incubated in media containingTelox or Telox2 (100 μM, added as a neat solution in sterile DMSO; final[DMSO]=0.1%) for 3 hours under atmosphere containing an appropriateconcentration of O₂. Following incubation, the media was removed, cellswere washed with sterile PBS, and then separated from the incubationplate via trypsinization (37° C., 10 min) and gentle scraping. The cellsuspension was pelleted and resuspended in PBS containing β-ME (1.0 mL,100 mM). Cells were pelleted, resuspended in PBS (900 μL, no β-ME), andfixed with formaldehyde (100 μL, 37% solution) for 25 minutes.

Following fixation, cells were pelleted, resuspended in PBS containingβ-ME (1.0 mL, 100 mM), and pelleted once again. The resultant pellet wasthen dissolved in ultra pure HNO₃ (1.0 mL, ˜35% solution) and half ofthe sample was submitted for analysis via ICP-MS. Signal for ¹³⁰Te wasthen normalized to signal for ¹¹⁵In at a known concentration (5 ppb,measured from ICPMS set-up solution, PerkinElmer) in order to accountfor detector sensitivity drift. The resultant signal was furthernormalized to give the sample with the maximum tellurium signal a valueof 1.0. All experiments were performed in duplicate.

vii. Mass Cytometry Experiments

HCT 116 cells (see sections i and ii) were incubated in media containingTelox or Telox2 (100 μM, added as a neat solution in sterile DMSO; final[DMSO]=0.1%) for 3 hours under atmosphere containing an appropriateconcentration of O₂. Following incubation, the media was removed, cellswere washed with sterile PBS, and then separated from the incubationplate via trypsinization (37° C., 10 min) and gentle scraping. The cellsuspension was pelleted and resuspended in PBS containing β-ME (1.0 mL,100 mM). Cells were pelleted, resuspended in PBS (900 μL, no β-ME), andfixed with formaldehyde (100 μL, 37% solution) for 25 minutes. Followingfixation, cells were pelleted, resuspended in PBS containing β-ME (1.0mL, 100 mM), and pelleted once again. Cells were then resuspended in PBS(990 μM, no β-ME) and incubated with either the Ir-containing nucleicacid intercalator (hypoxic cells) or the Rh containing nucleic acidintercalator (normoxic cells) (10 μL, 100 μM solution in sterile PBS)for 20 minutes. Cells were then pelleted and resuspended in PBS (1.0 mL,no β-ME) twice. Cell pellets were then resuspended in PBS containing^(151/53)Eu beads (1/10 dilution of CyTOF® calibration beads, DVSSciences, 1.0-2.5 mL depending on pellet size). The two cells sampleswere then combined (250 μL of each) and 250 μL of the resultant samplewas injected onto a second-generation CyTOF® instrument for MC analysis.For experiments involving Ir and Rh, cell samples were not mixedtogether, but rather, run separately on the CyTOF®. For the Pimonidazolecompetition experiment, cells were simultaneously incubated withPimonidazole and Telox or Telox2 (100 μM of each), with all other stepsexecuted in an identical manner to the experiment described above (Note:only the Ir-intercalator was used for this experiment since all sampleswere run separately).

Results and Discussion Methyl Telluroether: Synthesis and Stability

The organotellurium functionality that was initially investigated wasthe methyl telluroether due to its small size and ease of synthesis(Table 1, Compounds 1-4, 8-9). Aryl telluroethers were not investigateddue to numerous reports of their redox-activity in living systems andtheir reported instability under ambient light.¹⁹ The methyltelluroethers were synthesized from nucleophilic lithium methyltellurolate, using a modified procedure first established by N. Khun,followed by reaction with the desired nucleophile (Scheme 1).²⁰ Thesynthesis of compound 3 required quenching the methyl telluroate withwater to generate the tellurol prior to the Michael-style addition tomethyl acrylate. The yields of these additions ranged from 66 to 91%.

The relative chemical stability of the methyl telluroethers (1-4) werequantified using ¹H NMR by integration of the CH₃—Te signals withrespect to a residual DMSO-d₅ internal standard. Samples were preparedin a solution of DMSO-d₆ and placed under a slow continuous stream ofdry ambient atmosphere in a clear glass desiccator. This setup allowedthe compound's stability to be monitored without interference fromatmospheric water (FIG. 2).

Compounds 1-4 all degraded over the course of the 24 hr. incubation.Compound 4 was the most stable alkyl telluride investigated, degradingapproximately 15% over 24 hours (FIG. 2B). Compounds 2 (FIG. 2A) and 3(FIG. 2B) showed the greatest degradation, approximately 75% and 85%,respectively. (FIG. 2A).

The alkyl tellurides are presumed to undergo oxidation under theexperimental conditions. During incubation the initially yellowsolutions became colourless with the formation of white precipitate, atvarying rates. This phenomena has been previously observed and ispresumed to be the telluroxide species forming polymeric structures orthe formation of TeO₂.^(21&22) In addition, it has been observedpreviously that solutions of TelOx showed the appearance of ¹H NMRabsorptions consistent with chalcogen oxidation.^(5,23) Alkyl telluridecompounds are also known to undergo hemolytic bond cleavage and this mayresult in the observed small quantities of dimethyl ditelluride anddimethyl telluride.²⁴⁻²⁶ These species are volatile and are observedonly in the early time points of compounds 1 and 2. The same species areobserved in the 8 hour sample from compound 3. In addition, methylacrylamide was produced during the degradation of compound 3. Theremaining organic components resulting from these degradations could notbe identified and may be lost due to their low molecular weight andvolatility. To reduce the propensity for oxidative degradation of thetelluroethers, the trifluoromethyl telluroethers, and 10, and thetellurophenes 6, 7 and 11 were investigated.

Trifluoromethyl Telluroether: Synthesis and Stability

Compound 5 bearing a trifluoromethyl group will have reduced electrondensity at the tellurium center and thus should oxidize more slowly.Compound 5 was synthesized by the generation of tetramethylammoniumtrifluoromethyl tellurolate in situ by treating tellurium metal withtrimethyl(trifluoromethyl)silane and tetramethyl ammonium fluoride.²⁷Methyl-4-bromobutyrate was added to the solution to give the product 5in 50% yield (Scheme 1).

The stability of the trifluoromethyl telluride 5 was evaluated under thesame conditions as compounds 1-4 (FIG. 2B). Comparison of thestructurally related compounds 4 and 5, supported the hypothesis thatreducing electron density at the Te center would stabilize the compound,as no degradation was observed over the 24 hour incubation suggestingthe trifluoromethyl functionality was stabilizing the telluroether ashypothesized.

Tellurophene: Synthesis and Stability

Tellurophenes have not been evaluated in biological systems, and onlyrecently has the first water soluble tellurophene been reported.²⁸Tellurophenes possess interesting photophysical properties and have beeninvestigated as light harvesting agents for solar cell applications andin materials chemistry.^(29,30,31) The chalcogen analogue, selenophenes,have been investigated in biological systems with promise as antioxidantmolecules. Through computational analysis, ground state aromaticity oftellurophenes are considered to be more stabilized than selenophenes.³²It was hypothesized that the aromatic nature of the tellurophene wouldprovide greater chemical stability over the telluroether derivativesunder the desired biological conditions.

The tellurophenes where synthesized via the addition of Te²⁻ to amono-functionalized diacetylene in a synthesis modified from Stephensand Sweat's initial report.³³ ³⁴ In synthesizing these tellurophenes,the generation of Te²⁻, commonly, performed by treating an aqueoussuspension of Te⁰ with NaBH₄, was carried out using a basic Rongalite(NaHOCH₂SO₂) solution which reproducibly generated Te²⁻ and gave higheryields of the desired tellurophene³⁵

The trialkylsilyl diacetylenes can, for example, be generated inexcellent yield using the Cadiot-Chodkiewicz cross-coupling reaction(Scheme 2).³⁶ Bromination of triisopropylsilyl acetylene usingN-bromosuccinimide and silver nitrate gave the known couplingintermediate using the conditions of Wulff et al.³⁷ This compound wasthen coupled using CuCl in 30% BuNH₂ with the desired acetylenecomponent of choice. The synthesis of compound 6 utilized the startingmaterial 4-pentynoic acid, and compound 7 utilized the starting material3-butyn-1-ol. The trialkylsilyl protected diacetylene compounds weredeprotected using tetrabutylammonium fluoride and cyclized intotellurophenes 6 and 7 using a basic solution of Rongalite and telluriummetal in good yield (Scheme 2).

The stability of the two tellurophenes was studied using the sameprotocol as the alkyl telluride species (FIGS. 2 A and B). Compounds 6and 7 have improved stability in comparison to the methyl alkyltelluroethers. Both tellurophene compounds degraded insignificantly overthe 24 hr. incubation having similar stability to thetrifluoromethyltelluroether 5.

Benzylamine Coniuqated Organotellurium Derivatives: Synthesis andStability

For the future generation of MC probes, the organotelluriums areconjugated to biologically relevant functional groups. Here twoconjugation reactions were considered that provide a means to labelprimary amines, a carbamylation and an amidation reaction. Furthermore,forming benzylamine derivatives of the organotellurim compounds 2, 4, 5and 7, leads to compounds (8-11) with more comparable partitioncoefficients for cell toxicity studies (Schemes 3 & 4).

Compound 8 was synthesized via a p-nitrophenyl carbonate generated bythe treatment of compound 2 with p-nitrophenyl chloroformate. Thereactive carbonate could be purified and stored. Compounds 9 and 10 weresynthesized by hydrolysis of the methyl ester and, after isolation ofthe carboxylic acid, coupling proceeded with DCC in the presence of NHSand benzylamine. Compound 11 was synthesized analogously to 9 and 10 butcolumn chromatography was used for purification.

The stability of these benzylamine derivatives was assessed using thedeveloped ¹H NMR assay (FIG. 2C). The rate of degradation of thesecompounds mirrored those of the underivatized compounds with the methyltelluride species, compounds 8 and 9, degrading 20-25% over theincubation and compounds 10 and 11 being stable over the incubation.Interestingly the carbamate 8 was considerably more stable than theparent alcohol 2 suggesting the alcohol may directly contribute to thedegradation mechanism.

Stability in PBS Buffer

Of the compounds evaluated, the trifluoromethyltelluroether-amide (10)and the tellurophene-amide (11) exhibited the best stabilities underaerobic conditions. To further validate these compounds as potential MCmass tags, the degradation was also studied in a buffered aqueoussolution by dissolving the compounds in a 50/50 solution of d-DMSO/PBSbuffer. The compounds were kept in an environment exposed to air andambient lighting at room temperature. ¹⁹F NMR was used to study compound10 using a trifluoroacetic acid internal standard while ¹H NMR was usedto study the degradation compound 11 using the d5-DMSO internalstandard. As shown in FIG. 3, the trifluoromethyltelluroether 10 showeda 60% degradation after 24 hours. However, under the same conditions thetellurophene 11 was stable.

Cellular Toxicity

To investigate the organotellurium compounds of study as potential probemoieties for MC, the metabolic toxicity of the compounds was evaluated.Organotellurium compounds are often described as toxic, with aryltelluroethers showing cellular toxicity below 100 μM across a range ofcell lines under different assay conditions.^(9-11,38,39) The toxicityof compounds 8-11 was investigated in a commonly used Jurkat cell lineafter a 24 hour incubation using the metabolic probe WST-1 (RocheDiagnostics, Laval, Quebec) as per manufacturer's instructions. Ascompounds 8, 9 and 10 are expected to show degradation over the timeframe of the toxicity assay, based on the NMR stability studies, theseexperiments show the relative toxicity of the compounds and theirresulting degradation products (Table 2). Compound 8 had an apparentLD₅₀ value of 610 μM, but with a large experimental error due to thelack of solubility of compound 8 at higher concentrations. Compounds 9and 10 were more toxic with an LD₅₀<200 μM, however the tellurophene 11was less toxic with and LD₅₀ of 280 μM. These data suggest that, ingeneral, the alkyl telluroethers and the tellurophenes are less toxicthan previously investigated aryl telluroethers. The LD₅₀ values of theexemplary organotellurium compounds of the present application providepromise for their use as activity based MC probes since the general MCexperiment can be achieved at concentrations of ˜100 μM.⁵

Example 17

MC is a powerful analytical tool. Tellurium has valuable characteristicsas a mass tag for MC including eight stable isotopes, minimal functionalgroup size and minimal polarity. Various organotellurium compoundsfunctionalized for MC probe development have been synthesized andcharacterized. The alkyl telluride species are synthetically tractablebut have comparatively less compound stability. The tellurophene moietyis available in good yield, is chemically stable and is sufficientlynon-toxic.

Telox-2 takes advantage of the same 2-nitroimidazole functionality asTelox, however, the tellurium-containing mass tag is significantlydifferent. Instead of the methyltelluroether functionality in Telox,Telox-2 employs a tellurophene heterocycle (see the functionalitylabelled “mass tag” in Scheme 14).

To investigate the stability of Telox-2 compared to Telox ¹H NMR spectraof Telox-2 were collected over a period of 1 week underbiologically-relevant conditions (0.01% dDMSO in D₂O). Telox-2 exhibitsremarkable stability for a heavy chalcogen-containing molecule, as NMRdata suggests that no observable degradation occurs under the conditionstested (FIG. 5). Additionally, the lack of observable peak-broadening inthe ¹H NMR spectrum of Telox-2 at high concentrations (2 mM) suggeststhat Telox-2 does not form aggregates or micelles in aqueous solution.

An orthogonal stability assay using UV-Vis spectroscopy was performed tocorroborate the NMR stability findings (FIG. 6). In this assay, UV-Visspectra of Telox-2 were recorded at a fixed concentration in aqueousbuffer over a period of 3 days. Loss of either heterocycle, telluropheneor nitroimidazole, as a result of degradation would be expected to causea decrease in absorbance at 282 and/or 325 nm respectively. No change inabsorbance was detected in this experiment, thus corroborating our NMRfindings that Telox-2 is an exceptionally stable organochalcogen. Theempirical log P value of 1.3 for Telox-2 was measured using similarUV-Vis assay conditions. This log P value is lower than would bepredicted for the corresponding thiophene and partially explains theunusually high water solubility of Telox-2.

Next, the toxicity profile of Telox-2 was investigated using the sameassays that were employed for Telox. Confluency analysis (FIG. 7 part a)indicated that Telox-2 is begins to slow cell proliferation at aconcentration between 50 and 100 μM under normoxic and between 100 and200 μM under hypoxic conditions. This proliferative toxicity profile iscomparable to Telox, perhaps suggesting that the 2-nitroimidazolefunctionality is the limiting toxicity factor rather than thetellurium-containing functional group. Metabolic toxicity as measuredvia a WST-1 assay (FIG. 8 part b) indicated a metabolic LD₅₀ of 270 μMfor Telox-2; a value that is, once again, similar to Telox.

Oxygen labelling was investigated using the scheme in FIG. 4a . Resultsfor Telox are shown in FIGS. 4b-e ). Evaluation of oxygen-labelling anddose-labelling relationships for Telox-2 (FIG. 8) in HCT116 cellsrevealed an optimal probe concentration of 10 μM (under theseconditions) to maximize signal-to-noise (i.e. specificlabelling+nonspecific binding) between cells incubated under normoxicconditions vs. those incubated under near-anoxic conditions.Signal-to-noise for cells incubated under moderately hypoxic conditions(1% O₂) was significantly lower than for their near-anoxic counterparts.

The time-dependence of labelling under conditions of constant drugexposure was then evaluated using the optimized dose (FIG. 10 part a).This experiment demonstrated that signal-to-noise is enhanced asincubation time increases when comparing both near-anoxic and moderatelyhypoxic cells to the normoxic control. Although a useful experiment forin vitro assays, constant drug exposure conditions are unrealistic forin vivo experiments since drug clearance would be expected to rapidlyreduce the probe concentration in an animal model. In order to bettersimulate an in vivo scenario, the time-dependence of Telox-2 labellingafter pulsed exposure to the probe was investigated. In this experiment,cells were exposed to Telox-2 for a period of 3 hours, after whichprobe-containing media was removed and replaced with fresh media. Thecells were then allowed to incubate for (up to) an additional 21 hoursbefore MC analysis was performed (FIG. 9 parts c and d). The results ofthis experiment indicated that Telox-2-protein conjugates (see Telox-2Scheme part b) are lost according to an exponential-type decay (FIG. 9part c). This is consistent with expectations, as the amount ofTelox-2-protein conjugate per cell is expected to decrease with time inthe absence of additional un-metabolized Telox-2 as cells are expectedto divide over the time period investigated, thereby passing half of theTelox-2-protein conjugate to daughter cells after each division.

To confirm the reductive metabolism of Telox-2 it investigated usingmutant HCT116 cell lines that either overexpressed NADPH:cytochrome P450oxidoreductase (POR) or had this enzyme knocked out (FIG. 10 part b). Inthe cell line overexpressing POR, greatly enhanced labelling withTelox-2 (as compared to wild type cells) was observed, suggesting thatthis enzyme is important in the metabolism and subsequent activation ofTelox-2. Interestingly, Telox-2 labelling in the POR-knockout cell linewas nearly identical to that in the wild type (FIG. 10 part c). Thissuggests that cells are able to metabolize Telox-2 through alternativeoxidoreductase enzymes that may be upregulated in compensation for thelack of POR.

Under competitive conditions with pimonidazole, Telox-2 labelling wasreduced in a (pimonidazole) dose-dependent manner (FIG. 11). Completecompetition was not observed even at excessively high doses ofpimonidazole suggesting that a metabolic pathway exists that is capableof reducing Telox-2 but incapable of processing pimonidazole.Competition was only observed under near-anoxic conditions suggestingthat the small tellurium signal observed in cells incubated undernormoxic conditions is not a result of reductive metabolism.

Example 18 Cathepsin S

Cells are grown under normal conditions and/or one or more testconditions and incubated with a cathepsin S substrate labelled with atellurophene compound such as

wherein the DTPA portion of the molecule is labelled with a second metaltag (tag2), optionally a tellurophene moiety with a distinct mass. Thesubstrate associates with the membrane due to the compounds fatty acidtail. If Cathepsin S is active, the substrate is cleaved releasing thesoluble DTPA portion of the molecule. The ratio of tellurophene/tag2signal is indicative of cathepsin S activity. For example a 1:1 ratiowould be indicative that cathepsin was not active whereas a ratio ofless than one would be indicative of activity. These probes can besynthesized by those skilled in the art using schemes similar toliterature examples (e.g. Angew. Chem. Int. Ed. Engl. 53(29):7669-7673.doi:10.1002/anie.201310979 incorporated herein by reference).

Example 19 Enzyme Linked Assay

An alkaline phosphatase (AP) substrate comprising an organotellurophenetag, for example,

can be prepared using the following scheme

AP can be immobilized to a plate or bead directly or indirectly as partof an antibody conjugate (e.g. where an antibody conjugate has beenincubated with an antigen immobilized on a plate or bead). The telluriumtagged substrate, which generates a quinone methide upon phosphatecleavage, is incubated with the bead or well. If AP is present, thephosphate is cleaved and the quinone methide reacts with AP and/or otherbiomolecules in the vicinity. Tellurium levels are used as a readout ofthe presence and/or amount of AP present.

AP can be part of an antibody conjugate used for antigen detection on acell or tissue sample. The tellurium tagged substrate, which generates aquinone methide upon cleavage of the phosphate ester is incubated withthe cells or tissue sample. If AP is present the phosphate is cleavedand the quinone methide reacts with AP and/or other biomolecules in thevicinity. Tellurium levels are used as a readout of the presence and/oramount of AP present.

Example 20

Enzyme localization can be performed using tellurophene taggedcompounds. Tissue samples are incubated with a tellurophene taggedenzyme substrate that upon cleavage forms a precipitate comprising thetellurophene moiety. For example tellurophene linked 3,3′Diaminobenzidine is a substrate of horse radish peroxidase that producesan insoluble polymer upon reaction. The presence and amount of horseradish peroxidase would be indicated by Tellurium associated with theinsoluble polymer. In another example tellurophene bound to a2-(2′-phosphoryloxyphenyl)-6-[125I]iodo-4-(3H)-quinazolinone wouldprovide a water soluble alkaline phosphatase substrate which releases aninsoluble tellurium linked quinazoline which precipitates locally uponphosphate ester cleavage. The amount of alkaline phosphatase activitywould correlate with the insoluble tellurium compound formed. Imagingmethods are used to detect the tellurium precipitate.

Tables

TABLE 1 Organotellurium compounds investigated

1

2

3

4

5

6

7

8

9

10

11

TABLE 2 LD₅₀ values of the organotellurium compounds 8-11. Compound LD₅₀(μM) 8  610 ± 290* 9 180 ± 60 10 130 ± 20 11 280 ± 30 *Compound 8 has alarge experimental error due to the lack of solubility. All cells couldnot be killed at the maximal concentration acceptable for the experiment(2 mM).

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely

While the present application has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the application is not limited to the disclosedexamples. To the contrary, the application is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Specifically, the sequences associated with eachaccession numbers provided herein including for example accessionnumbers and/or biomarker sequences (e.g. protein and/or nucleic acid)provided in the Tables or elsewhere, are incorporated by reference inits entirely.

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1. A compound of formula (I):

wherein A is a naturally occurring isotope of Te; R¹ is selected from H,unsubstituted or substituted C₁-C₂₀alkyl, unsubstituted or substitutedC₃-C₂₀cycloalkyl, unsubstituted or substituted aryl and an electronwithdrawing group; L is C₁₋₃₀alkylene, unsubstituted or substituted withone or more substituents, and/or optionally interrupted with one or moreheteromoieties independently selected from O, S, NR⁷, and/or optionallyinterrupted with one or more of C(O) and C(S); R⁷ is independentlyselected from H, PG and C₁₋₆alkyl; X is a reactive functional groupselected from halo, OH, OTs, OMs, C(O)H, C(O)OR⁸, C(O)NR⁹R¹⁰,O—C(O)—OR¹¹, O—C(O)—NR¹², C(O)ONR¹³R¹⁴, C(O)R¹⁵, C(O)SR¹⁶ and NR¹⁷R¹⁸;R⁸ is selected from H, C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, wherein thelatter three groups are unsubstituted or substituted with one or more ofhalo and NO₂; R⁹ and R¹⁰ are independently selected from H, C₁₋₆alkyl,aryl and C₁₋₆alkylenearyl, wherein the latter three groups areunsubstituted or substituted with one or more of halo and NO₂, or R⁹ andR¹⁰, together with the N atom to which they are bonded, form a 4 to 12membered monocyclic or bicyclic, saturated or unsaturated ringunsubstituted or substituted with one or more ═O, ═S, halo andC₁₋₆alkyl; R¹¹ is selected from C₁₋₆alkyl, aryl, and C₁₋₆alkylenearyl,wherein the latter three groups are unsubstituted or substituted withone or more of halo and NO₂; R¹² is selected from C₁₋₆alkyl, aryl, andC₁₋₆alkylenearyl, wherein the latter three groups are unsubstituted orsubstituted with one or more of halo and NO₂; R¹³ and R¹⁴ areindependently selected from H, C₁₋₆alkyl, aryl and C₁₋₆alkylenearyl,wherein the latter three groups are unsubstituted or substituted withone or more of halo and NO₂, or R¹³ and R¹⁴, together with the N atom towhich they are bonded, form a 4 to 12 membered monocyclic or bicyclicsaturated or unsaturated ring unsubstituted or substituted with one ormore ═O, ═S, halo and C₁₋₆alkyl; R¹⁵ is halo; R¹⁶ is selected fromC₁₋₆alkyl, aryl and C₁₋₆alkylenearyl, wherein the latter three groupsare unsubstituted or substituted with one or more of halo and NO₂ R¹⁷and R¹⁸ are independently selected from H, C(O)C₁₋₆ alkyl, C₁₋₆alkyl,aryl and C₁₋₆alkylenearyl, wherein the latter three groups areunsubstituted or substituted with one or more of halo and NO₂, or R¹⁶and R¹⁷, together with the N atom to which they are bonded, form a 4 to12 membered monocyclic or bicyclic saturated or unsaturated ringunsubstituted or substituted with one or more ═O, ═S, halo andC₁₋₆alkyl; and one or more available hydrogens are optionally replacedwith D; or a salt and/or solvate thereof; with proviso that when X isC(O)OR¹⁹ and R¹⁹ is H or C₁₋₆alkyl, L is not C₁₋₈alkylene; and when X isOH, L is not C₁₋₂alkylene. 2.-7. (canceled)
 8. The compound of claim 1,wherein L is a C₁₋₂₅alkylene, unsubstituted or substituted with one ormore substituents independently selected from C₁₋₃alkyl, C(O)R⁴ andNR⁵R⁶, and/or optionally interrupted with one or more heteromoietiesindependently selected from O and NR⁷, and/or optionally interruptedwith C(O); R⁴ is selected from H and C₁₋₂alkyl; R⁵ and R⁶ areindependently selected from H, PG, C(O)C₁₋₆alkyl and C(O)OC₁₋₆alkyl; andR⁷ is independently selected from H and PG.
 9. The compound of claim 8,wherein L is a C₁₋₂₅alkylene, unsubstituted or substituted with one ormore substituents independently selected from NR⁵R⁶, and/or optionallyinterrupted with one or more heteromoieties independently selected fromO and NR⁷, and/or optionally interrupted with C(O); R⁵ and R⁶ areindependently selected from H, PG, and C(O)OC₁₋₄alkyl; and R⁷ is H.10.-12. (canceled)
 13. A compound of formula (II):

wherein A is a naturally occurring isotope of Te; R¹ is selected from H,unsubstituted or substituted C₁-C₂₀alkyl, unsubstituted or substitutedC₃-C₂₀cycloalkyl, unsubstituted or substituted aryl and an electronwithdrawing group; L is C₁₋₂₀alkylene, unsubstituted or substituted withone or more substituents, and/or optionally interrupted with one or moreheteromoieties independently selected from O, S, NR⁷, and/or optionallyinterrupted with one or more of C(O) and C(S); R⁷ is independentlyselected from H, PG and C₁₋₆ alkyl; and Z is a biosensor, biologicallyactive material, or polymeric backbone; and/or a salt and/or solvatethereof. 14.-19. (canceled)
 20. The compound of claim 13, wherein L is aC₁₋₂₅alkylene, unsubstituted or substituted with one or moresubstituents independently selected from C₁₋₃alkyl, C(O)R⁴ and NR⁵R⁶,and/or optionally interrupted with one or more heteromoietiesindependently selected from O and NR⁷, and/or optionally interruptedwith C(O); R⁴ is selected from H and C₁₋₂alkyl; R⁵ and R⁶ areindependently selected from H, PG, C(O)C₁₋₆alkyl and C(O)OC₁₋₆alkyl; andR⁷ is independently selected from H and PG.
 21. The compound of claim20, wherein L is a C₁₋₂₅alkylene, unsubstituted or substituted with oneor more substituents independently selected from NR⁵R⁶, and/oroptionally interrupted with one or more heteromoieties independentlyselected from O and NR⁷, and/or optionally interrupted with C(O); R⁵ andR⁶ are independently selected from H, PG, and C(O)OC₁₋₄alkyl; and R⁷ isH.
 22. The compound of claim 13 wherein Z is a biosensor.
 23. Thecompound of claim 22, wherein the biosensor comprises 2-nitroimidazole.24. (canceled)
 25. The compound of claim 13, wherein Z is a biologicallyactive material.
 26. (canceled)
 27. (canceled)
 28. The compound of claim13 wherein Z is a monomeric unit of a polymeric backbone and thecompound comprises at least one of formula (IIa):

wherein n is an integer representing the number of repeating monomericunits of formula (IIa). 29.-32. (canceled)
 33. A compound of claim 25,wherein the biologically active material is selected from an affinityreagent selected from an antibody or binding fragment thereof, aptamer,avidin reagent, nucleic acid or lectin. 34.-65. (canceled)
 66. A kit forperforming a mass detection assay, the kit comprising: a plurality oforganotellurophene compounds; wherein each compound comprises a distincttellurium isotope; and wherein each compound comprises a biosensor,biologically active compound, polymeric backbone, or a reactivefunctional group for tagging a biosensor, biologically active compound,or polymeric backbone.
 67. The kit of claim 66, wherein one or more ofthe compounds comprise a biosensor.
 68. The kit of claim 67, wherein thebiosensor is an enzyme substrate.
 69. The kit of claim 66, wherein oneor more of the compounds comprise a biologically active material,wherein the biologically active material is an affinity reagent.
 70. Thekit of claim 69, wherein the affinity reagent is an antibody.
 71. Thekit of claim 67, wherein one or more of the compounds comprise apolymeric backbone.
 72. The kit of claim 67, wherein one or more of thecompounds comprises a reactive functional group, wherein the reactivefunctional group is maleimide.
 73. The kit of claim 67, wherein one ormore of the compounds comprises a tellurophene heterocycle.
 74. The kitof claim 67, wherein the compounds are packaged for use as barcodingreagents.